Towards a potent and rapidly reversible Dexmedetomidine-based general anesthetic

Zheng Xie; Robert Fong; Aaron P Fox

doi:10.1371/journal.pone.0291827

. 2023 Sep 26;18(9):e0291827. doi: 10.1371/journal.pone.0291827

Towards a potent and rapidly reversible Dexmedetomidine-based general anesthetic

Zheng Xie ^1,^*, Robert Fong ¹, Aaron P Fox ²

Editor: Silvia Fiorelli³

PMCID: PMC10522005 PMID: 37751454

Abstract

Clinically useful anesthetics are associated with delirium and cognitive decline in the elderly. Dexmedetomidine (Dex), an α₂ adrenergic receptor agonist, is an intravenous sedative with analgesic properties. Dex is associated with a lower incidence of delirium in the elderly. In this study, we first assessed whether a high dose of Dex alone was a clinically useful anesthetic. Finding that it was not, we sought to determine whether supplementation of Dex with low doses of two common anesthetics, propofol or sevoflurane, created an effective general anesthetic. Rats were sedated with a bolus followed by a continuous infusion of Dex and a low dose of a second agent—propofol, or sevoflurane. A strong noxious stimulus was applied every 15 minutes while monitoring vital signs. A combination of the α₂ competitive antagonist, atipamezole, and caffeine was administered to reverse the anesthesia. Abdominal surgery was used to validate the efficacy of these dosing regimens. The animals responded to noxious stimuli when receiving Dex alone. Supplementing Dex with either a low dose of propofol or sevoflurane completely suppressed responses to the noxious stimulus and allowed the rats to tolerate abdominal surgery with complete immobility and no alterations in vital signs, suggesting that the drug combinations were effective anesthetics. EEG recordings showed suppression of high frequency activity suggesting that awareness and memory were impaired. Previously we found that combination of atipamezole and caffeine rapidly and completely reversed the sedation and bradycardia elicited by Dex. In this study, atipamezole and caffeine accelerated the time to emergence from unconsciousness by >95% in Dex supplemented with either propofol or sevoflurane.

In conclusion

Our results suggest that Dex supplemented with a low dose of a second agent creates a potent anesthetic that is rapidly reversed by atipamezole and caffeine.

Introduction

Anesthesia in current clinical practice is exceptionally safe [1]. As such, anesthesia research has focused on optimizing currently available anesthetics rather than developing new agents [1]. Many of the anesthetics currently in use were developed decades ago [2–8]. Despite their favorable safety profile, these anesthetics, including sevoflurane and propofol, are associated with delirium and cognitive dysfunction in the elderly [9–13]. Additionally, neuroapoptosis and cognitive alterations were observed in animal studies that included non-human primates [14–20]. Although unambiguous evidence of neurotoxicity in humans remains to be demonstrated, the search for new and potentially safer anesthetic regimens represents a valuable endeavor. Dexmedetomidine (Dex) is a sedative associated with a lower incidence of delirium and cognitive complications in the elderly [10, 21–23] and neural protection in the young [24, 25]. Our goal in this study was to determine whether Dex alone, at high dosages, could produce an effective anesthetic. If Dex alone was not an effective anesthetic, then we sought to determine whether supplementing Dex with low, subanesthetic doses of two common anesthetics, propofol or sevoflurane, produced an efficacious intraoperative general anesthetic.

A successful anesthetic embodies four cardinal traits; amnesia, unconsciousness, antinociception and immobility [26]. Amnesia is difficult to study in animal models, while unconsciousness, antinociception and immobility are readily quantified. While Dex is an effective sedative, it is a poor amnestic and immobilizer at the concentrations used clinically.

First, high dose Dex was tested to determine whether rats remained unconscious, unresponsive and immobile when exposed to a powerful noxious stimulus. Next, low doses of two common anesthetics, propofol and sevoflurane, were used to supplement Dex. Although propofol and sevoflurane have been implicated in neurotoxicity, high doses of the anesthetics were required to initiate apoptosis and cognitive decline in young animals. The low doses of these anesthetics employed in this study fall below the range shown to engender these deleterious effects. Furthermore, Dex seems to mitigate apoptosis caused by other anesthetics [27, 28], suggesting that Dex may be neuroprotective [29–31]. Future neuroapoptosis and behavioral studies will be required to confirm the safety of the drug combinations used in our study.

Both drug combinations, Dex/propofol or Dex/sevoflurane, suppressed responses to a powerful noxious stimulus, while high dose Dex alone did not. Furthermore, Dex with sevoflurane or propofol suppressed all motor and autonomic responses during abdominal surgery. Propofol, and sevoflurane are amnestic at low concentrations [32–34]: Our EEG recordings suggest that memory is impaired by the anesthetic combinations we tested. Dex with low-dose sevoflurane or propofol produced less burst suppression than sevoflurane alone near its EC₅₀.

We have shown previously that a combination of low dose atipamezole and caffeine reverses Dex sedation with remarkable effectiveness [35]. This reversal cocktail accelerated emergence from unconsciousness produced by Dex supplemented with propofol or sevoflurane with equal efficacy. Our results suggest that these drug combinations based primarily on Dex meet the requirements for an effective general anesthetic that is rapidly reversed by low dose atipamezole and caffeine.

Materials and methods

Ethics and animals

This animal study was approved by The University of Chicago Institutional Animal Care and Use Committees (protocol #42437). This manuscript adheres to the applicable ARRIVE guidelines. Between experiments animals were cared for by University of Chicago veterinary staff. Forty female Adult Sprague Dawley rats (Charles River, Wilmington, MA), weighing 250–400 gm and 8 male rats weighing 250–350 grams were used in the study. They were transported to the anesthesia room for multiple anesthesia sessions with at least five days in between sessions. At the completion of each anesthesia session, rats were transported back to their own home room. Rats were divided into groups of 8 for each set of experiments. Each rat was never sedated more than 6 times. All rats served as their own controls. All experiments were performed during the daytime and at room temperature of 22–27°C. While on a nose cone and throughout the study rats were placed on a heating pad at 25°C. During experiments, heart rate, respiratory rate and blood oxygen saturation were monitored with a Kent Scientific PhysioSuite. SpO₂ was always >92% throughout the experiments. In 2 subsets of experiments, blood pressures (BP) from the tails of the rats were measured by a BP system (IITC Life Science Inc., CA). Noxious stimuli were stopped at the first sign of distress. At the conclusions of the study, rats were euthanized by the animal facility staffs using CO₂ overdose, followed by decapitation. In experiments where surgery was performed, the rats were sacrificed, after the wound were closed with sutures, by an overdose of propofol (20 mg/kg) and decapitation by veterinary staff.

Calibrated noxious stimulus

A calibrated tail clamp was used to assess anesthetic efficacy. The stimulus was generated with Kelly forceps that were used to clamp each rat’s tail where it was exactly 5 mm in diameter. Clamping the tail to the first stop on the forceps for 30 seconds, or until a response was evoked, provided a consistently reproducible stimulus. Elevated levels of sevoflurane was required to suppress motor response to the tail clamp.

Determination of minimum alveolar concentration (MAC) equivalence of sevoflurane in suppressing tail clamp stimulus

Minimum alveolar concentration (MAC) is used to quantify the potency of inhaled anesthetics, representing the EC₅₀ for motor response to a noxious stimulus [36, 37]. The rats were initially anesthetized by placement into a gas-tight chamber (volume 6 liters) into which sevoflurane was delivered by an anesthesia machine (Ohmeda Modulus II Plus). A nose cone was connected to the outlet port of the gas chamber by a short length of corrugated tubing. The gas concentration was sampled at the outlet port of the gas chamber by a gas analyzer (Intellivue MP70, Philips). After each rat was anesthetized in the gas chamber with 3.3% sevoflurane with 2LO₂/2LAir for 10 minutes, the rat was weighed and then placed with its face in the nosecone. A 24g intravenous catheter was placed in a tail vein and the rat was anesthetized with 3% sevoflurane with 1LO₂/1LAir via the nose cone for another 20 minutes to reach steady state. The initial anesthetic concentration was chosen as 1.2–1.3 MAC as determined by other studies [38–40]. The tail clamp was applied as described above. Depending on the rat’s response to the tail clamp, sevoflurane was dialed up for positive response or down for negative response by 0.3%. The rat was anesthetized with the next concentration of sevoflurane for 15 minutes before repeating the tail clamp. The test would end if the rat stopped responding to the tail clamp on the step up or responded to the tail clamp on the step down. The concentration where 50% of rats did not respond to the tail clamp was considered the MAC for sevoflurane.

Drugs

Caffeine (Sigma-Aldrich, St Louis, part # C0750-5G, Lot#SLBD0505V) was dissolved in sterile saline to a final concentration of 10 mg/ml, and rats were dosed intravenously to a final dose of 25mg/kg. Sterile saline injection was used as vehicle control for caffeine.

Atipamezole (also called Antisedan) was manufactured by Zoetis Pharmaceuticals, Parsippany, NJ (#RXANTISEDAN-10). The same bottle was used for the entire study. The atipamezole dosages used in the studies outlined in the manuscript varied from 5 μg/kg to 20 μg/kg. Atipamezole was administered intravenously to the rats with sterile saline as the vehicle at 5 μg/ml.

Dexmedetomidine (Dexmedetomidine hydrochloride: NDC 16729-239-93) was purchased from Accord Healthcare, Durham, NC, at a 5 μg/ml concentration in saline. Dex was delivered intravenously by an infusion pump (Medfusion 4000, Smith Medical ASD, Inc. St. Paul, MN).

Propofol (Diprivan injectable emulsion NDC 63323-269-29) was purchased from Fresenius (Kabi, Lake Zurich, IL) at a 10 mg/ml concentration. The drug was delivered by an infusion pump.

Sevoflurane (Ultane, NDC 0074-4456-04) was purchased from Abbvie, North Chicago, IL.

Sedation/ Anesthesia

Rats were placed in a gas-tight anesthesia chamber where they were exposed to either 1.8% isoflurane or 3.3% sevoflurane (in 2L/min O₂ & 2L/min Air) for 10 minutes, rendering them unconscious and insensitive to mild tail pinch. Rats were then removed from the gas tight chamber and weighed. Anesthesia was maintained with 1.8% isoflurane or 3.3% sevoflurane in 1L/ min O₂ & 1 L/min Air, delivered via a nose cone. A 24g intravenous (IV) catheter was inserted into a tail vein. Isoflurane was used to render rats’ unconscious such that they could be weighed and an I-V line inserted. In some experiments, EEG electrodes were inserted, and the EEG recorded under isoflurane was used as a baseline. Isoflurane was terminated after the bolus dose of Dex or Dex with propofol was delivered. The tail clamp stimulus was applied only after the washout of isoflurane was complete. In sevoflurane experiments we used sevoflurane throughout. The IV was inserted after 20 minutes 3.3% sevoflurane, followed by 1.4% sevoflurane with Dex bolus and infusion.

Dex infusion with and without a second agent

Dex was administered via an infusion pump attached to the IV line. A bolus of Dex was delivered over 5 minutes via a pump. This was followed by 60 minutes of continuous infusion of Dex (see Fig 1). Every 15 minutes following the bolus, vital signs were recorded, and the response to tail clamp assessed. The rats breathed 1L/min O₂ & 1 L/min Air for the entire duration of Dex exposure.

Fig 1 — For this protocol, a 5-minute infusion of a bolus of Dex (10 μg/kg) was followed by 60 minutes of a maintenance infusion of Dex (10 μg/kg/hr), left panel. The same rats were exposed to a similar protocol, but one where a second agent supplemented the Dex, either propofol or sevoflurane, right panel. The second agent was present for the entire Dex infusion. At various times, the rats were tested with a tail clamp to measure immobility. Vital signs were obtained throughout the experiment. At the end of the protocol, rats received an injection of either saline or atipamezole with caffeine (randomized order). Rats were then placed on their backs in a waking box, and the time for the rats to recover their righting reflex was recorded. This time is plotted in the subsequent Figs as the emergence time.

The second lumen of a Y shaped microcatheter (Baxter Healthcare Corp, Deerfield, IL) allowed us to use a second infusion pump for propofol. Reversal agents or saline were injected by syringe connected directly to the IV catheter and flushed with 0.5 ml saline. The IV catheter was then removed, and the rats were placed in a cage on their backs. The time to emergence was defined as the time required for the rats to right and stand on all 4 paws. (Also referred to as recovery of righting reflex–RORR).

Electroencephalogram (EEG) recording

Scalp electrodes were used for electroencephalographic recording. To avoid stress effects following invasive surgical implantation of electrodes, we employed 9 mm stainless steel EEG needle electrodes inserted into the scalp during anesthesia so that they were touching the outer table of the skull. After rats were anesthetized with 3.3% sevoflurane or 1.8% isoflurane, two scalp electrodes [Astro-Med/ Grass Technologies] were placed, as shown in S1 Fig. We drew a line between the anterior edge of bilateral ears, between Bregma and Lambda as described in reference [41]. From the midpoint, one electrode was placed anteriorly perpendicular to the line and the other posteriorly perpendicular to it. Two EEG channels were recorded, the first one (red) from an electrode placed over the anterior portion of the brain, and a second electrode (green) placed over the posterior portion of the brain. The EMG lead (yellow) was obtained from an electrode placed over the left shoulder, all referenced to an electrode (white) placed near medial to the ears. A ground electrode (black) was placed on the opposite side of the reference lead. These three channels were recorded with an A/D rate of 500 Hz/channel, with a 0.05–100 Hz bandpass and 12 dB/octave roll-off. Potentials were amplified with a Neuroscan SYNAMPS 2 system (Compumedics, Inc., Charlotte, NC).

Power spectra

Two types of power spectra were computed: conventional power spectra using the SYNAMP EDIT module, and spectrograms using MATLAB R2021, and the EEGLAB program, using time resolution of 10 seconds with 99% overlap. Power spectra (dB) were computed over 5-minute-long epoch of EEG, partitioned into 512-point epochs, and averaged, yielding a temporal resolution of 2 Hz. Power was calculated as the fraction of a specific frequency power, including delta (0.5–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), spindle (12–15 Hz) and beta (15–25 Hz) by MATLAB R2021. The average powers of 8 rats in each frequency band were compared in the different period of times between sevoflurane 3.0% sevoflurane and 1.4% sevoflurane with Dex infusion in one group and between 1.7% isoflurane and propofol and Dex infusion in another group. Burst suppression ratio (BSR) in the EEG was calculated by a formula, BSR = (total time of suppression/epoch length) x 100% and analyzed independently by two different members of this study. Each independent analysis produced consistent results. Suppression time was defined from 0.5 to 5 seconds consistent with other studies [42–44]. Burst suppression in EEG was defined as an amplitude < 5 μV which lasted for ≥ 30% of each minute.

Power spectra were obtained for two 5-minute periods and reference two separate conditions during the session. In one group, animals were exposed to sevoflurane 3.0%, then sevoflurane was turned down to 1.4%, then Dex bolus10 μg/kg and infusion 12 μg/kg/hr started. Power spectra were obtained under each condition, including after forty minutes of the subanesthetic dose of sevoflurane together with Dex infusion. In another group, the animals were first exposed to isoflurane 1.7% for 25 minutes, then isoflurane was turned off and washed out, and propofol 4 mg/kg plus Dex bolus10 μg/kg were given over 5 minutes and followed by propofol 300 μg/kg/min and Dex 12 μg/kg/hr infusion. Power spectra were obtained under each condition including forty minutes into subanesthetic dose of propofol with Dex infusion. During these anesthesia sessions, the global changes in EEGs were more prominent anteriorly. Therefore, we used the signal recorded from the anterior lead for analysis in this study.

Statistical analysis

The sample size used in this study was based on an analysis described in a previous study from our lab and by using GPower [35]. In this study the threshold for statistical significance was set to 0.05. The statistical test used to analyze each data set is described in the appropriate figure legends. If three or more comparisons were required within a group of animals a repeated measures analysis of variance (RM-ANOVA) with Tukey’s multiple comparisons post-hoc test was employed. Data was evaluated for normality. When only 2 conditions were assessed, either a paired or an unpaired T-test was employed Data was analyzed and graphed using GraphPad Prism 9 software. Data were expressed and plotted graphically as mean ± standard deviation (SD).

The experiments shown in this manuscript were done in an unblinded manner. Experimental order and drug application were randomized.

All data obtained in this study were shown in this manuscript and supporting information.

Results

Is high dose of Dex an effective anesthetic?

To assess whether high doses of Dex suppressed responses to a tail clamp, we subjected 8 rats on separate days to either a high dose or low dose infusion regimen. In the high dose regimen, we administered a 40 μg/ kg bolus of Dex over 5 minutes followed by a continuous infusion of Dex at 48 μg/ kg/ hr for an additional 60 minutes. In the low dose regimen, we administered a 10 μg/ kg bolus over 5 mins followed by a continuous infusion of Dex at 12 μg/kg/hr for an additional 60 minutes.

In animal studies of anesthetic efficacy, ablation of motor response to a noxious stimulus is typically assessed. Such a stimulus was generated by tail clamping as described in the Methods section. Every 15 minutes a painful tail clamp was applied to the rats, while monitoring the animals for any responses including vital sign changes. S1 Table compares the responsiveness to the tail clamp at various timepoints with these two dosing regimens in female rats. Aggregating the data from all 4 time points, S1 Table shows that there were only two positive responses to the tail clamp out of 32 total tests when animals received high dose Dex, compared to 25 positive responses out of 32 total tests when those same animals received low dose Dex (p<0.0001, Fisher’s exact test).

In a previous study we observed that after a single bolus of Dex at 40 μg/kg, rats required approximately 100 mins to regain their righting reflex as compared to 30 mins after a 10 μg/kg Dex bolus [35]. The infusion used in the current study should produce similar extended emergence times. Fig 2 shows that atipamezole and caffeine reversed the unconsciousness engendered by high dose Dex. While the rats required 10,891 ± 5928 seconds to emerge from unconsciousness following a control saline injection administered after terminating the high dose Dex infusion, they required only 17 ± 11.24 seconds to emerge after injection of 20 μg/ kg of atipamezole and 25 mg/ kg caffeine (p = 0.013, paired t-test). Although the rats recovered their righting reflex, they remained sluggish for an extended period. Thus, reversal was incomplete.

Fig 2 — The same group of 8 rats were exposed to two Dex’s sessions, a week apart. At the end of one session the rats received a bolus injection of saline and in the other atipamezole (20 μg/kg) and caffeine (25 mg/kg). The order of the drug injections was randomized. Rats were placed on their backs in a waking box, and the time for the rats to recover their righting reflex was recorded. Saline injected rats are plotted on the left while atipamezole and caffeine injected rats are plotted on the right. The >99% difference in emergence time was significant (p = 0.013, two tailed paired T-test, t = 5.196 and df = 7).

High dose Dex infusion was associated with a decrease in heart rate and respiratory rate, while blood oxygen saturation remained unchanged (Fig 3). Using a 2-way repeated measures ANOVA we found no difference in vital signs when comparing high dose to low dose Dex regimens in the same animals at the same time points. This result suggests that the cardiovascular changes observed following the administration of Dex saturate at the lower dosage.

Fig 3 — The vital signs were measured at various times during the experiment. The first measurement (“1.7% isoflurane”) took place while the rats were receiving 1.7% isoflurane, prior to Dex administration. The second measurement (“1.1% isoflurane”) was taken while the rats were receiving 1.1% isoflurane, prior to Dex administration. The third measurement (“End of Bolus”) was taken immediately after the bolus of Dex was administered. The 1.1% isoflurane was turned off but not yet washed out. Rats were breathing O₂. The fourth measurement (“t = 15”) was taken fifteen minutes after the end of the bolus. All isoflurane should have been washed out since rats had been breathing O₂ for 15 minutes. The fifth, sixth and seventh measurements (“t = 30”, “t = 45”, “t = 60”) were taken thirty, forty-five and sixty minutes after the end of the bolus. The green panels represent high dose Dex (bolus 40 μg/kg: infusion of 48 μg/kg/hr) while the blue panel data was obtained from the same rats exposed to a lower dose of Dex (bolus 10 μg/kg: infusion 12 μg/ kg/ hr). Comparisons of HR at different times for Dex 10/12. Dex 40/48 was similar. For this analysis a repeated measures two-way ANOVA was employed: 1.7% isoflurane vs. 1.1% isoflurane, p = ns: We compared the HR at 1.1% isoflurane to the rest of the time points, 1.1% isoflurane vs. End of Bolus, p = 0.002: 1.1% isoflurane vs. t = 15, p = 0.002: 1.1% isoflurane vs. t = 30, p <0.0001: 1.1% isoflurane vs. t = 45, p < 0.0001: 1.1% isoflurane vs. t = 60, p <0.0001. Comparisons of RR: Only the following times were different. 1.1% isoflurane vs. End of Bolus, p = 0.0003: End of bolus vs t = 15, p = 0.01: End of bolus vs t = 30, p = 0.01: End of bolus vs t = 45, p = 0.004: End of bolus vs t = 60, p = 0.02. Comparisons of SpO₂: No significant changes were observed. We found no evidence for a time by condition interaction (Dex 10/12 and Dex 40/48). Subsequent low Dex versus high Dex at each time point comparisons gave the following: no significant difference in HR for any time points: 1.7% isoflurane, 1.1% isoflurane, End of bolus, t = 15, t = 30, t = 45 and t = 60 between the two conditions with adjusted p = 0.64, p = 0.71, p = 0.99, p = 0.99, p = 0.99, p = 0.99 and p = 0.99, respectively. There was also no evidence for a time by condition interactions between the two conditions in RR and SpO2 at any time points.

After administration of the Dex boluses, HR was significantly lower for all subsequent times compared to that observed prior to application of Dex. The RR was significantly depressed by Dex, but just for a single time point. The RR recovered by 30 minutes after the Dex bolus and remained back at baseline levels for subsequent measurements. There was no change in SPO₂ elicited by the application of Dex.

Dex and low dose propofol

The goal of these studies was to determine whether a low dose of a second agent in combination with Dex produced an effective anesthetic. We define a “low dose” of the second agent as one incapable of maintaining unconsciousness as assessed by the ablation of the righting reflex. Rats received an intravenous bolus of 5 mg/kg of propofol over 5 minutes followed by a continuous infusion at varying concentrations for an additional 60 minutes. S2 Fig shows the time to emergence at various infusion doses of propofol. As is evident in S2 Fig, a minimum infusion dosing of 400 μg/kg/min was needed to maintain unconsciousness. At 300 μg/kg/min, all animals spontaneously emerged from anesthesia prior to the cessation of the hour-long infusion. Because these propofol levels were sub hypnotic, 300 g/kg/min or less, they were considered “low dose”.

S2 Table shows the results of an experiment in which a low dose of propofol was used to supplement Dex. Rats were first anesthetized with isoflurane (1.8%) to allow intravenous cannulation. They then received a bolus of both Dex (10 μg/ kg) and of propofol (4 mg/ kg) over 5 minutes, after which the isoflurane was terminated, and the rats breathed an O₂/Air mixture. Over the next 60 minutes, the animals received infusions of Dex (15 μg/ kg/ hr) and propofol (200 μg/ kg/ min). We applied a tail clamp every 15 minutes to the rats while recording vital signs. At the end of the hour the infusions were discontinued, and the animals were injected with either saline (control) or with atipamezole (20 μg/ kg) and caffeine (25 mg/ kg). We then placed the rats on their backs to assess the time for recovering their righting reflex.

S2 Table shows that aggregating the data from all 4 time points, that for Dex alone the rats responded to the tail clamp 24 out of 32 times tested, compared to only 3 out of 64 times for Dex and low dose propofol (p<0.0001, Fisher’s Exact Test). This dose of propofol when combined with Dex was insufficient to completely suppress responses to the tail clamp.

We initially tested Dex at a dose we employed in our previous study [35] with propofol at 200 μg/kg/min. This dose was not able to ablate tail clamp response in all animals tested. Therefore, we repeated the experiment with a higher dose of propofol and slightly lower dose of Dex.

When the experiment was repeated with an infusion of 300 μg/kg/min of propofol to supplement the Dex at 12 μg/kg/hr, none of the rats responded to the tail clamp at any time point (0 responses out of 32 tests, S3A Table). This difference was significantly different than Dex by itself (p < 0.0001, n = 32). From the data shown above, 300 μg/kg/min represents the lowest possible dose of propofol to supplement Dex if the goal is to achieve complete suppression of any response to the tail clamp. These results suggest that a Dex infusion of 12 μg/kg/hr with a second agent is sufficient to produce an effective immobilizing and antinociceptive agent. Therefore, Dex infusion at 12 μg/kg/hr was used as the base in subsequent Dex combinations in the present studies.

Repeating the same experiment with a cohort of 8 male rats showed an identical result. When Dex was used by itself, the rats responded to the tail clamp 31 times out of 32 total tests (S3B Table). When Dex was supplemented with the low dose of propofol (4 mg/ kg bolus, then infusion of 300 μg/kg/min) none of the rats responded to the tail clamp at any time point. Reponses to the tail clamp in Dex alone were significantly different than those in Dex supplemented with low dose propofol (p < 0.0001, Fisher’s exact test).

Prolonged emergence time has been a significant drawback to the use of Dex infusions in clinical practice. Previously we showed that atipamezole and caffeine effectively reversed sedation engendered by Dex. In this experiment we tested whether atipamezole and caffeine could reverse the combination of Dex and low dose propofol. Fig 4 shows that it took the female rats 3284 ± 786 (mean ± SD) seconds to emerge from unconsciousness when they received a control saline injection, but a mere 141.9 ± 123.2 seconds to emerge after injection of atipamezole (20 μg/ kg) and caffeine (25 mg/ kg) a >95% difference which was significant (p < 0.0001, two tailed paired T-test, t = 11.89 and df = 7).

Fig 3 shows that atipamezole (10 μg/kg) & caffeine (25 mg/kg) were equally effective at accelerating emergence in male rats exposed to Dex & propofol (4 mg bolus/ 300 μg/kg/min infusion). Emergence was rapid, occurring at105.4 ± 75.4 sec (mean ± SD). We previously showed that male rats woke in ~2700 seconds for this dose of Dex when used by itself [35].

The combination of Dex and propofol used to generate the data in S3 Table and Fig 4 was associated with a statistically significant decrease in heart rate and respiratory rate, while blood oxygen saturation remained unchanged (Fig 5). Vital signs recorded with Dex alone were not different from those recorded in Dex with propofol.

Fig 5 — The vital signs were measured at various times during the experiment. The first measurement (“1.7% isoflurane”) took place while the rats were receiving 1.7% isoflurane, prior to Dex administration. The second measurement (“1.1% isoflurane”) was taken while the rats were receiving 1.1% isoflurane, prior to Dex administration. The third measurement (“End of Bolus”) was taken immediately after the bolus of Dex was administered. The 1.1% isoflurane was turned off but not yet washed out. Rats were breathing O₂/Air. The fourth measurement (“t = 15”) was taken fifteen minutes after the end of the bolus. All isoflurane should have been washed out since rats had been breathing O₂/Air for 15 minutes. The fifth, sixth and seventh measurements (“t = 30”, “t = 45”, “t = 60”) were taken thirty, forty-five and sixty minutes after the end of the bolus. Statistics: A one-way repeated measures ANOVA was performed to compare the effects of Dex at different time points with that before Dex application. Tukey’s HSD Test for Multiple Comparisons found that the mean values for HR were significantly different in the presence and absence of Dex. Comparisons of HR: 1.7% isoflurane vs. 1.1% isoflurane, p = 0.44: We compared the HR at 1.1% isoflurane to the rest of the time points, 1.1% isoflurane vs. End of Bolus, p <0.0001: 1.1% isoflurane vs. t = 15, p <0.0001: 1.1% isoflurane vs. t = 30, p <0.0001: 1.1% isoflurane vs. t = 45, p < 0.0001: 1.1% isoflurane vs. t = 60, p <0.0001. Comparisons of RR: 1.1% isoflurane vs. End of Bolus, p <0.0001: 1.1% isoflurane vs. t = 15, p <0.0001: 1.1% isoflurane vs. t = 30, p <0.0001: 1.1% isoflurane vs. t = 45, p <0.0001: 1.1% isoflurane vs. t = 60, p <0.0001. Comparisons of SpO₂: 1.7% isoflurane vs. 1.1% isoflurane, p = 0.95: We compared the HR at 1.1% isoflurane to the rest of the time points, 1.1% isoflurane vs. t = 15, p <0.06: 1.1% isoflurane vs. t = 30, p = 0.41: 1.1% isoflurane vs. t = 45, p = 0.43: 1.1% isoflurane vs. t = 60, p = 0.28.

Determination of EC50 or minimum alveolar concentration (MAC) equivalence of sevoflurane

The minimum alveolar concentration (MAC) is defined as the concentration of an inhalational anesthetic agent that suppresses response to a noxious stimulus in half of a test population [36, 45]. In published studies, 2.0% - 2.4% sevoflurane was identified as ~1 MAC in adult rats [38, 39]. S4 Table show that in our study, 3.0% sevoflurane was required to suppress response to tail clamping in 50% of rats tested and thus represents 1 MAC. The most likely explanation for the difference in MAC between our results and that in the literature is that we measured gas concentrations in the corrugated tubing leading to the rat’s nose cone and not alveolar levels. Previous studies have shown that measurement differences give rise to significantly different MAC values. For example, MAC values using anesthesia box gas values were different than those measured assaying alveolar gas concentrations at the tracheotomy site [46].

Dex and low dose sevoflurane

Dex was next paired with 1.4% of sevoflurane (< 0.5 MAC), considered to be “low dose” based on our experimental determination of a MAC of 3% as outlined above. At this dose of sevoflurane alone, most rats would move or right themselves with a mild stimulus such as IV insertion. Further reducing sevoflurane dose might lead to the recovery of righting reflex (RORR) in some rats before the completion of Dex bolus. As before, a bolus of Dex (10 μg/kg) given over 5 minutes, followed by a continuous infusion of Dex (12 μg/kg/hour) for the next 60 minutes.

S3 Table shows that aggregating data in female rats from all 4 time points, no animal responded to the tail clamp in when Dex was paired with low-dose sevoflurane, 32 total tests, while in rats treated with Dex alone, 25 responded in 32 total tests (P<0.0001).

In female rats, Atipamezole (10 μg/ kg) and caffeine (25 mg/ kg) rapidly reversed Dex alone (9.5 ± 10.99 sec, n = 8) as well as the combination of Dex and sevoflurane (146.3 ± 46.60 sec, n = 8), although it was more effective at reversing Dex by itself (p < 0.0001). Without reversal, rats took 3584 ± 903.9 (mean ± SD) seconds to emerge from Dex and sevoflurane anesthesia (Fig 6). Atipamezole and caffeine was equally effective at accelerating emergence in male rats receiving Dex supplemented with sevoflurane, taking a mere 69.9 ± 23 sec (S3 Fig) for emergence to take place.

The combination of Dex and low-dose sevoflurane used to generate S3 Table and Fig 6 in female rats was associated with a statistically significant drop in heart rate and respiratory rate, while blood oxygen saturation remained unchanged (Fig 7). Vital signs recorded in Dex by itself were not different from those recorded in Dex with sevoflurane. Male rats were similar (S4 Fig).

Fig 7 — The vital signs were measured at various times during the experiment. The first measurement took place while the rats were receiving (“3.0% sevoflurane”), prior to Dex administration. The second measurement (“1.4% sevoflurane”) was taken while the rats were receiving 1.4% sevoflurane, prior to Dex administration. The third measurement (“End of Bolus”) was taken immediately after the bolus of Dex was administered. The 1.4% isoflurane was turned off but not yet washed out. Rats were breathing O₂. The fourth measurement (“t = 15”) was taken fifteen minutes after the end of the bolus. All sevoflurane should have been washed out since rats had been breathing O₂ for 15 minutes. The fifth, sixth and seventh measurements (“t = 30”, “t = 45”, “t = 60”) were taken thirty, forty-five and sixty minutes after the end of the bolus. Statistics: A one-way repeated measures ANOVA was performed to compare the effects of Dex at various times. Tukey’s HSD Test for Multiple Comparisons found that the mean values for HR were significantly different in the presence and absence of Dex. Comparisons of HR: 3.0% sevoflurane vs. 1.4% sevoflurane, p = 0.21: We compared the HR at 1.4% sevoflurane to the rest of the time points, 1.4% sevoflurane vs. End of Bolus, p = 0.0012: 1.4% sevoflurane vs. t = 15, p = 0.0003: 1.4% sevoflurane vs. t = 30, p = 0.0001: 1.4% sevoflurane vs. t = 45, p < 0.0001: 1.4% sevoflurane vs. t = 60, p = 0.0001. Comparisons of RR: 1.4% sevoflurane vs. End of Bolus, p = 0.001: 1.4% sevoflurane vs. t = 15, p = 0.053: 1.4% sevoflurane vs. t = 30, p = 0.03: 1.4% sevoflurane vs. t = 45, p = 0.03: 1.4% sevoflurane vs. t = 60, p = 0.019. Comparisons of SpO₂: 3.0% sevoflurane vs. 1.4% sevoflurane, p = 0.98: We compared the HR at 1.4% sevoflurane to the rest of the time points, 1.4% sevoflurane vs. End of Bolus, = 0.99: 1.4% sevoflurane vs. t = 15, p >0.99: 1.4% sevoflurane vs. t = 30, p = 0.99: 1.4% sevoflurane vs. t = 45, p = 0.99: 1.4% sevoflurane vs. t = 60, p >0.99.

Abdominal surgery

To assess whether Dex in combination with a low dose of another anesthetic could provide robust general anesthesia, we performed abdominal surgery involving incision of the skin, underlying abdominal muscles and the peritoneum on groups of 4 rats (see Fig 8). Immobility during surgery represents the gold standard for successful anesthesia [26]. These rats were anesthetized with either Dex in combination with 1.4% sevoflurane, or Dex in combination with propofol (4 mg/kg bolus followed by continuous infusion of 300 μg/kg/min). None of the animals undergoing this surgical procedure exhibited any motor or autonomic response. Fig 8A showed an illustration of the abdominal incision in one rat. Fig 8B plots heart rate, respiratory rate, SpO₂ and mean arterial pressure immediately before and after skin incision for Dex and propofol anesthesia. Vital signs were unchanged by incision.

Fig 8 — A, shows the surgery that took place in one rat. First there was an incision through the skin. Then the abdominal muscles were cut. Finally, the peritoneum was perforated exposing the abdominal cavity. At no stage of the surgery did the rats respond by moving or by a change in vital signs. B, the combination of Dex and low-dose propofol produced a powerful anesthetic that prevented movement or change in vital signs while surgery was performed. A bolus of Dex (10 μg/kg) was followed by continuous infusion of Dex (12 μg/kg/hr). Propofol was applied as a 4 mg/kg bolus followed by a continuous infusion of 300 μg/kg/min. The skin was cut ~18 minutes after finishing the bolus. Plotted are pairs of measurements taken just before skin incision and just after. A line connects the two points. Heart rate, respiration rate, SpO₂ and mean arterial pressure are plotted for each rat, in this group of 4.

Taken together, the results of these experiments suggest that both combinations tested, Dex with propofol or Dex with sevoflurane, represent potent anesthetics, producing deep levels of unconsciousness, immobility and antinociception.

EEG analysis: Sevoflurane vs Dex with low sevoflurane, isoflurane vs Dex with low propofol

Dex sdoes not produce a reliable amnestic effect when used by itself at the cllinical doses [47]. Sevoflurane and isoflurane, by contrast, manifest strong amnestic effects with an extremely low incidence of intraoperative awareness or recall in humans [48, 49]. We recorded EEG activities near 1 MAC of sevoflurane or isoflurane, conditions in which awareness and recall are very unlikely, to serve as a baseline for comparison to Dex infusion with low dose sevoflurane (<0.5 MAC) or low dose propofol (300 μg/kg/min). Fig 9A and 9B provide an example of a raw EEG trace and 5-minute spectrogram obtained from a rat anesthetized with 3% sevoflurane. Fig 9C and 9D depicts the raw EEG trace and 5-minute spectrogram obtained in the same rat receiving 1.4% sevoflurane with Dex (12 μg/kg/hr).

Fig 9E compares the power spectra from two 5-minute epochs; one in 3% sevoflurane and the other in 1.4% sevoflurane with Dex (12 mg/kg/hr) averaged from a group of 8 rats. Under both anesthetic conditions, delta bands (0.5–4 Hz) predominated in the power spectrum and were not different between the two periods (p = 0.87, n = 8). The powers in frequency bands of theta (4–8 Hz), alpha (8–12 Hz), spindle (12–15 Hz), and beta (15–25 Hz) were higher under sevoflurane 3% than under sevoflurane 1.4% with Dex (p = 0.003 **, p = 0.002 **, p< 0.000 and p = 0.0008 **, respectively).

Fig 9F plots the burst-suppression ratio over two 5-minute periods, one under sevoflurane 3% (Mean ± SD, 0.79 ± 0.01, n = 8) and the other under sevoflurane 1.4% with Dex infusion (Mean ± SD, 0.18 ± 0.10, n = 8). Sevoflurane 3% produced a higher BSR than did sevoflurane 1.4% with Dex (p <0.0001, n = 8).

Fig 10A and 10B provide an example of a raw EEG trace and 5-minute spectrogram during 1.7% isoflurane anesthesia from one rat. Fig 10C and 10D depicts the raw EEG trace and 5-minute spectrogram from the same rat receiving Dex 12 μg/kg/hr with propofol 300 μg/kg/min infusion.

Fig 10E compares power spectra of 5-minute epochs; one taken from 1.7% isoflurane and propofol 300 μg/kg/min with Dex 12 mg/kg/hr infusion from a group of 8 rats. Under both anesthetic conditions, the delta bands (0.5–4 Hz) accounted for the bulk of the power and were not different between the two periods (p = 0.78, n = 8). Alpha bands (8–12 Hz) were the same with both anesthetic conditions (p = 0.32). The powers in theta (4–8 Hz), spindle (12–15), and beta (15–25) frequency bands were higher under isoflurane 1.7% than under propofol with Dex (p = 0.017, p< 0.0001 **** and p = 0.0002 ***, respectively).

Fig 10F plots the burst suppression ratio over two 5-minute periods, one under 1.7% isoflurane (Mean ± SD, 0.57 ± 0.04, n = 8) and the other under propofol 300 μg/kg/min with Dex 12 mg/kg/hr infusion (Mean ± SD, 0.02 ± 0.01, n = 8). Isoflurane 1.7% caused a higher burst suppression ratio than did low dose propofol with Dex (p<0.0001, n = 8).

No adverse events occurred in the rats throughout this study.

Discussion

Dex is associated with less postoperative delirium and neurocognitive dysfunction in the elderly [50, 51]. Dex is also associated with less neuroapoptosis and cognitive alterations in developing brains of various animal models, including non–human primates [24, 27, 29, 52–54]. Dex is common in pediatric sedation and as an adjunctive agent in general anesthesia. It is reasonable to posit that using Dex as the primary agent in an anesthetic regimen would maximize its beneficial effects. Unfortunately, Dex is neither a powerful immobilizer nor an amnestic agent. Dex has other drawbacks including prolonged unconsciousness following sedation as well as bradycardia and hypotension. Bradycardia leading to hypotension is usually overcome by fluid bolus in pediatric sedation or fluid and glycopyrrolate in adults. Without a reversal agent available, prolonged recovery times are common.

Atipamezole is a selective α₂ receptor antagonist. While it has been shown to reverse the effects of Dex in humans, high doses (Atipamezole: Dex ratio of 40–100:1) were required, which were associated with unwanted effects including emesis, motor restlessness, and increased blood pressure (>20 mm Hg) [55–58]. Due to this unfavorable side effect profile, atipamezole has not been approved for use in humans. For veterinary medicine, the manufacturer recommends an Atipamezole: Dex ratio of 10:1 for rapid reversal [59]. A study in rats from our lab paired low dose atipamezole with caffeine; atipamezole was used at a dose too low to engender adverse effects (atipamezole: Dex ratio 1:1) [35]. Together, the atipamezole with caffeine were remarkably effective and emergence times decreased by ~97% compared to control [35]. Low dose atipamezole and caffeine may represent a clinically useful reversal cocktail for Dex based anesthetics. The absence of such a reversal cocktail has thus far limited the wider use of dexmedetomidine.

In this study, we first evaluated high dose Dex to see if it could produce unconsciousness and immobility. Next, we assessed whether Dex supplemented with a low dose of propofol, or sevoflurane produced unconsciousness and immobility. The secondary agents were used at dosages too low to produce anesthesia or even unconsciousness by themselves. Reversal of anesthesia by atipamezole and caffeine was assessed.

We administered a 10 μg/kg bolus of Dex, followed by either 12 μg/kg/hr or 15 μg/kg/hr infusion for 60 minutes. All rats required ~1 hour to recover with no reversal agent. Dex induced unconsciousness with 100% efficiency but did not reliably produce immobility when tested with a tail clamp, suggesting that these doses of Dex did not produce anesthesia. Increasing the dose of Dex (40 μg/kg bolus, 48 μg/kg/hr infusion for 60 minutes) almost completely suppressed the motor response to the tail clamp (30/32 tests) but also prolonged emergence time by ~3-fold. Interestingly, high dose Dex did not engender further depression of respiratory rate or heart rate beyond that produced by lower doses of Dex, suggesting that the hemodynamic effects saturate. High dose Dex was reversed by atipamezole and caffeine (>99% reduction in emergence time). However, the rats remained sluggish with minimal movement for another 30 to 60 minutes after initial righting, suggesting that high dose Dex is not completely reversible. These results are consistent with reports of surgery carried out with high dose Dex alone in human patients [60].

Previous studies have shown that when Dex was used as an adjunct agent, it reduced the MAC concentration of isoflurane, sevoflurane and other volatile agents in rats and humans [52, 61]. Several clinical trials currently underway are exploring the use of Dex with other anesthetics or sedatives to maximize clinical benefit and minimize side effects [61–64]. Dex was used as an adjunct at doses ranging from 0.3–0.7 mcg/kg/hr in these studies. At these doses, Dex alone is not able to cause unconsciousness in humans subjects, but can reduce the MAC concentration of sevoflurane by 20–30%. When Dex was used at a higher dose alone, it prolonged recovery significantly [60]. Without a clinically effective reversal for Dex, the use of higher doses of Dex with or without a second agent is not practical. With the success of using low dose atipamezole and caffeine combination to reverse Dex effectively,we are able to employ anesthetic combinations in which Dex serves as the primary agent, with only small subanesthetic doses of another agent as an adjunct to enable Dex to function as a robust general anesthetic. We characterize Dex as the “primary” agent in our anesthetic combinations because it is employed at a dose that produces greater “anesthetizing power” than the dose of the other agent with which we combine it. This dex dose alone is able to maintain unconsciousness in rats throughout the duration of its infusion, while both the low dose sevoflurane and propofol doses we used in our combination regimens are unable to do so when used alone. Our anesthetic combinations represent an alternative and corollary approach that emphasizes Dex as the central component and employs a minimal dose of a second agent in an adjunctive capacity. With this strategy, the Dex doses evaluated in our study are nevertheless closer to the doses used in humans than the ones used in Veterinary Medicine. Dex doses up to 3 μg/kg/hr are commonly used for pediatric procedural sedation, such as that used for MRI scans [65]. In contrast, Dex 0.5–0.75 mg/kg IP is used in combination with ketamine at 75–150 mg/kg IP to anesthetize small animals, like rats, for surgery (see for example - https://animalcare.ubc.ca/sites/default/files/documents/Guideline%20-%20Rodent%20Anesthesia%20Analgesia%20Formulary%20%282016%29.pdf).

Infusing propofol (300 μg/kg/min) in combination with Dex (12 μg/kg/hr) completely suppressed any responses to the tail clamp and surgery in all animals tested. Dex and propofol together appear to recapitulate all aspects of an effective general anesthetic. Moreover, the unconsciousness produced by Dex and propofol was rapidly reversed by low dose atipamezole with caffeine (Fig 4).

Combining Dex with 1.4% sevoflurane (<0.5 MAC) produced unconsciousness and complete immobility in response to tail clamp and surgery. This drug combination was also rapidly reversed by a low dose of atipamezole with caffeine (Fig 6).

The combination of atipamezole and caffeine is as effective for reversing Dex with propofol or Dex with sevoflurane as it is in reversing Dex by itself [35]. This effective reversal provides a solution for the slow emergence of Dex based anesthetics. This may increase the potential for clinical use of Dex based anesthetics.

While it is difficult to directly assess awareness or amnesia in rats, the question can be addressed indirectly. Firstly, while Dex is not an effective amnestic when used alone, the agents we paired them with have been shown to be highly effective amnestic agents on their own at the dosages we employed [33, 66, 67]. Secondly, the lack of motor response to the tail clamp as well as trans-peritoneal abdominal incision suggests that an adequate depth of anesthesia was achieved. Finally, data obtained from EEG recordings of our anesthetized animals suggested that memory was impaired based on the similarities of EEG patterns produced by ~ 1 MAC sevoflurane or isoflurane and our Dex combination regimens. We compared the EEG activity recorded under low dose sevoflurane with Dex or low dose propofol with Dex to 1 MAC of sevoflurane or isoflurane alone. With all these anesthetic regimens, the power spectra were similar, characterized by dominance of delta frequency bands. Slow waves (delta band) are consistently present in the surgical phase of general anesthesia [68]. Notably, low dose sevoflurane with Dex and low dose propofol with Dex produced very low burst suppression ratios while sevoflurane or isoflurane near MAC levels produced much higher burst suppression ratios. Studies suggest that prolonged burst suppression under general anesthesia may be associated with higher incidences of post-operative delirium and neurocognitive dysfunction in elderly patients [69]. Therefore, avoiding lengthy periods of burst suppression may benefit vulnerable populations.

Using Dex as a primary anesthetic supplemented with a subanesthetic dose of second agent may represent a more receptor specific anesthetic strategy which limits unwanted pleiotropic effects. Inhalational agents produce their anesthetic effects, unconsciousness and immobility, by interacting with a variety of receptors, ion channels and second messenger systems within neural circuits [26, 70]. The sites of anesthetic actions of unconsciousness and immobility are in the brain and spinal cord, respectively. Despite years of research, a comprehensive mechanistic understanding of the anesthetic state remains elusive [71]. In vitro, inhalational or intravenous anesthetic agents produce their effects at high concentrations [72]. At these high concentrations, anesthetics interact promiscuously with multiple targets and may result in untoward effects including nausea, respiratory depression, myocardial suppression, and immunomodulation.

By contrast, Dex is the most selective agent currently used for either sedation or anesthesia, exerting its effects at nanomolar concentrations in vitro [73, 74]. Dex selectively activates α₂ receptors, which activates the G_i/o signaling pathway resulting in the inhibition of adenylate cyclase, thereby lowering intracellular cAMP levels [75, 76]. Activation of this pathway also activates GIRK-K⁺ channels and inhibits voltage gated Ca²⁺ channels. Activation of the G_i/o pathway inhibits neuronal activity and decreases neurotransmitter release.

Caffeine increases [cAMP]_i levels by inhibiting phosphodiesterase, which breaks down cAMP, thereby directly countering an important cellular effect of Dex. We predict that other stimulants which elevate [cAMP]_i should be equally effective. Low dose atipamezole and caffeine seem to work synergistically to reverse Dex mediated sedation.

A recent study showed that a large dose of amphetamine, a potent stimulant with a half-life of ~10 hours, could rapidly reverse Dex by itself [77]. A combination of low dose atipamezole with a lower dose of amphetamine may be able to reverse dexmedetomidine rapidly while minimizing the prolonged effects of the stimulant.

The ability to rapidly reverse sedation engendered by even high doses of dexmedetomidine overcomes one of the major barriers to its routine application in general anesthetic regimens. This study represents an incremental advance toward realizing the goal of receptor targeted anesthesia articulated by Talke in his 1998 editorial [78]. Our goal is to create Dex based anesthetic combinations that are even more specifically receptor targeted, and to extend these strategies into clinical studies.

Limitations

Plasma concentrations of Dex were not measured. Human studies are needed to correlate the Dex concentrations in plasma to the anesthetic effects. In these studies, EEG leads were placed after rats were already anesthetized. Therefore, there was no baseline EEG before anesthesia. The drug combinations demonstrated strong antinociceptive effects during surgeries. Whether Dex supplemented with a second agent will reduce the use of opioids intraoperatively and minimize side effects postoperatively remains to be determined. Which of the anesthetic combinations proves to be “safest” in terms of neuroapoptosis in neonatal animal studies or in ameliorating cognitive decline or emergence delirium in the elderly remails to be determined. Studies in “aged” rats will provide useful insights into the safety of the Dex based drug combinations.

In summary, our study demonstrates that Dex supplemented with a low dose of either propofol or sevoflurane creates a potent anesthetic with a favorable safety profile that can be rapidly reversed by low dose atipamezole with caffeine. Translating these observations to human populations represents a high priority.

Supporting information

S1 Checklist. The ARRIVE guidelines 2.0: Author checklist.

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S1 Fig. Scalp EEG lead placement.

Two scalp electrodes were placed, as shown. We drew a line between the anterior edge of bilateral ears, between Bregma and Lambda. From the midpoint, one electrode was placed anteriorly perpendicular to the line and the other posteriorly perpendicular to it. Two EEG channels were recorded, the first one (red) from an electrode placed over the anterior portion of the brain, and a second electrode (green) placed over the posterior portion of the brain. The EMG lead (yellow) was obtained from an electrode placed over the left shoulder, all referenced to an electrode (white) placed near medial to the ears. A ground electrode (black) was placed on the opposite side of the reference lead. Signals recorded from the anterior lead were analyzed for global changes during anesthesia.

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S2 Fig. Determining the dose of propofol required to maintain unconsciousness to define a low dose of the drug.

For this experiment a group of rats received a bolus of 5 mg/kg of propofol, applied in 5 minutes via a pump, after which they received a continuous infusion of propofol, at different concentrations for an additional 60 minutes. All rats remained unconscious for the 60-minute infusion if the infusion rates of propofol were kept at or above 400 μg/kg/min. In contrast, 300 μg/kg/min was not sufficient to keep the rats unconscious during the infusion.

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S3 Fig. The combination of atipamezole and caffeine dramatically accelerated emergence from anesthesia produced by Dex alone or from the combinations of Dex with Sevoflurane or Dex with Propofol, in male rats.

The same group of 8 rats were exposed to three sedation sessions, a week apart. At the end of each session the rats received a bolus injection of atipamezole (10 μg/kg) and caffeine (25 mg/kg). Rats were placed on their backs in a waking box, and the time for the rats to recover their righting reflex was recorded. Data (RORR Times in seconds): Dex alone—5, 9, 5, 1, 29, 1, 15, 2, Dex with Sevoflurane—110, 72, 81, 59, 62, 32, 85, 59, Dex with Propofol—222, 182, 150, 44, 32, 59, 129, 25.

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S4 Fig. Male rats exposed to Dex alone or supplemented with a low dose of propofol or Dex supplemented with a low dose of sevoflurane exhibited slowed heart and respiration rates while leaving blood oxygen saturation unaffected.

Dex or Dex supplemented with either propofol or sevoflurane was applied at time = 0. Vital signs were then measured every 15 minutes. Comparisons of HR at different times for Dex 10/12. Dex/sevoflurane and Dex/propofol were similar and are not presented. For this analysis a repeated measures two-way ANOVA was employed: 1.7% isoflurane vs. 1.1% isoflurane, p = ns: We compared the HR at 1.1% isoflurane to the rest of the time points. 1.1% isoflurane vs. End of Bolus, p <0.0001: 1.1% isoflurane vs. t = 15, p = 0.0001: 1.1% isoflurane vs. t = 30, p <0.0001: 1.1% isoflurane vs. t = 45, p < 0.0001: 1.1% isoflurane vs. t = 60, p <0.0001, Comparisons of RR: Only the following times were different. 1.1% isoflurane vs. End of Bolus, p < 0.0001: End of bolus vs t = 30, p = 0.0006: End of bolus vs t = 45, p = 0.0001: End of bolus vs t = 60, p < 0.0001: Comparisons of SpO₂: No significant changes were observed.

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S1 Table. Comparing responses to a noxious stimulus in female rats exposed to a lower dose of Dex alone with rats administered a higher dose of Dex alone.

A noxious stimulus was applied at different time points in an experiment. Responses were tabulated and are presented numerically in the table. Statistical difference is calculated with Fisher’s exact test.

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S2 Table. Comparing responses to a noxious stimulus in rats exposed to Dex alone with rats administered Dex and a low dose of propofol.

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S3 Table. Comparing responses to a noxious stimulus in rats exposed to Dex alone with rats administered Dex and a low dose of either sevoflurane or propofol.

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S4 Table. A—determining the concentration of sevoflurane required to prevent half of the responses to a noxious mechanical stimulus.

Details about the stimulus are in the Methods. The green column represents ~1 MAC equivalence concentration or EC₅₀.

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Acknowledgments

We would like to thank the support from the Department and Anesthesia and Critical Care at the University of Chicago. We thank Chuanhong Liao, MS, the Biostatistics lab, Department of Public Health Science, the University of Chicago, for her help with the statistics. We also thank Dr, Vernon L. Towle, Professor, Department of Neurology, University of Chicago, for his support and advice on EEG recording and analysis.

Abbreviations

Dex: dexmedetomidine
Ati: atipamezole

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study is supported by a NIH grant (GM-116119) To ZX and APF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0291827.r001

Decision Letter 0

Silvia Fiorelli

23 May 2023

PONE-D-23-07486Towards A Potent and Rapidly Reversible Dexmedetomidine-Based General AnestheticPLOS ONE

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Reviewer #1: In Manuscript PONE-D-23-07486, Xie and colleagues present their research examining the effect of adding dexmedetomidine (DEX) with other general anesthetics, in order to create a more readily reversible anesthetic. Overall, the authors are to be commended on the amount of data presented in this manuscript, however, this large volume of data obscures issues with the design of their experiments. There are almost three separate papers and hypotheses tested. The experimental design would have been better served if the author concentrated on one general anesthetic (e.g., sevoflurane) and performed more complete experiments. Overall, the results are confusing as presented. It appears that the authors wanted to test whether the addition of DEX to a standard general anesthetic can reduce the dose of general anesthetic required. However, the authors tested four different general anesthetics using a model for anesthetic effects (loss of tail clamp response) and recovery (recovery of righting reflex – RORR). This model was then further examined using a surgical model (abdominal incision).

Due to the large amount of data, their message is confusing to discern. The paper would be better suited for publication if the paper was presented as a single general anesthetic study that was more complete. A subsequent paper could build from these findings, and further examine other general anesthetics. Specific comments are below.

1. The authors wished to examine if a low or high dose of DEX could enhance a general anesthetic’s effects, thus decreasing the dose (MAC) of anesthetic used. The authors first determined the MAC of isoflurane and sevoflurane. Interestingly, the authors found that their MAC was higher than those published previously by others using a rat model and explain the discrepancy as being due to the tail clamp stimulus.

Tables 1A/1B should be moved to Supplemental Data. It is background information that could be moved to decrease the size of the paper.

2. In a similar manner, the authors determined a “low dose” and “high dose” of propofol, but they did not use the tail clamp test, instead they used “consciousness.” Why did they not use the tail clamp test to be more consistent?

3. The data is hard to read as written. For example, page 14 (Paragraph starting with “Table 2”) presents a lot of data with associated p values, and it just doesn’t read well. This issue can be found throughout the results section, and I would suggest that these sections be rewritten without so much data. Also, why did the authors study 200 and also 300 mcg/kg/hr of propofol if 300 mcg/kg/hr was considered to be low dose. In essence, two “low doses” of propofol were studied.

4. The authors also wanted to see if DEX could be reversed using a commonly known and utilized antagonist, atipamezole. This drug has been used extensively in the veterinary community for reversal of DEX or its non-racemic mixture medetomidine. Thus I am not sure why the authors present atipamezole as a new agent. It may not work as well in humans (as they mention in their discussion), but it is not novel in my opinion.

If the authors wish to study reversal of DEX effects, it should not have been included in the same study as examining “low dose” propofol. This idea should have been tested separately.

5. The paper would have been better organized by studying one general anesthetic completely, without reversal. Then, they could have added a section examining DEX reversal from a general anesthetic. Similarly, the abdominal surgery portion of the study, could have been done with a single general anesthetic agent, not four general anesthetics.

6. Low and high dose DEX data (Figure 10 and Table 5) should be presented first, as the showing this effect should have been the initial study done before examining combinations of DEX with general anesthetics.

7. The data on using DEX combined with propofol, isoflurane, and sevoflurane for abdominal surgery does follow their prior data using a tail clamp, both are painful stimuli, but is the lack of change in vital signs proof of a complete anesthetic? This data could be removed without affecting their interpretation.

8. The EEG analysis is quite complex, and it appears that it is included to demonstrate that the DEX/general anesthetic combinations effect memory and awareness. Again, this data could be a separate paper and by combining it with their other data, the resulting message/interpretation is confused. Why did they feel the need to demonstrate these effects?

Again, the combination of Tail clamp experiments with four different general anesthetics, abdominal surgery, and EEG analyses seems unnecessary to demonstrate that DEX can enhance lower dose general anesthetics to produce an “anesthetized” state.

9. The protocols used seem a bit random. For example, rats were anesthetized with isoflurane prior to surgery required for measuring EEG, then DEX was given, and the animals given propofol boluses and then an infusion. Various drugs were given after 30 or 60 minutes (why these timepoints?), infusion doses were changed (e.g., DEX was dropped from 15 mcg/kg/hr to 12 mcg/kg/hr) but why?

10. The Discussion describes a lot of prior work on DEX reversal agents, which doesn’t appear to be the major point of the manuscript and has been previously published by this laboratory (Ref. 42). Again, this focus on DEX reversal takes away from their findings.

11. The authors state that they wish to examine if combining DEX with general anesthetics can allow for “sub-therapeutic” doses of the general anesthetic. In fact, other published work has already demonstrated that DEX can reduce the MAC of volatile agents in animals and humans. If this fact is known, then what does this study add to the literature? They state they their desire is to determine if DEX can be used as the primary anesthetic, but when combining it with other agents, which one is primary and which one is secondary? Or does it matter? I feel that the authors are arguing a fine point that is not as important.

The question is can one reduce the amount of general anesthetic needed (whether it is isoflurane, sevoflurane, propofol, or nitrous oxide) when combined with DEX. Again, this fact has been demonstrated so what does this study add besides a more thorough examination of multiple drug combinations?

12. The authors discuss a major limitation of their study, simply put, they only studied female rats. It has been shown that female rats are more sensitive to DEX effects than male rats. This issue seriously limits the interpretation of their data.

What the authors do not discuss completely in my opinion is why their data demonstrates such a major effect of DEX compared to prior work.

Reviewer #2: This is a feasibility study in rodents to test whether combining dexmedetomidine with low doses of conventional anesthetics is sufficient to provide surgical anesthesia. The rationale for the study is that conventional anesthetics are known to cause delirium and cognitive dysfunction in elderly patients, and dexmedetomidine is known to be less deleterious. The manuscript is well written, and the results are described clearly. However, I have several concerns that need to be addressed.

• The short title, “A strategy for creating a new anesthetic,” is misleading. “New anesthetic” implies a novel drug, but the authors describe a novel dosing regimen using existing anesthetics. This should be revised.

• As stated in the Abstract and Introduction, the premise of the study is that dex administration will allow for lower doses of conventional anesthetics that are associated with delirium and cognitive dysfunction in the elderly. However, the authors did not use aged animals and did not test for delirium or cognitive dysfunction in their study.

• A significant portion of the Introduction discusses the methods, results, and conclusions of the study. This content belongs in the Methods, Results, and Discussion sections, respectively. The Introduction should focus on the background and rationale.

• Why were only female rats used for the study? The NIH and most journals now require the use of both sexes to account for sex as a biological variable.

• Were the anesthetic exposures conducted in random order?

• It should be clearly stated in the manuscript that atipamezole is not approved for human use. This greatly limits the translational potential of these results to the clinical setting.

• There are far too many figures. Many of them should be combined.

• I find it curious that MAC values for sevoflurane and isoflurane were much higher than reported values in the literature. The authors attribute this to their tail clamp being a more potent noxious stimulus, but are other explanations possible? Were the vaporizers and agent analyzers properly calibrated? Maybe the equilibration times were too short?

• In the Discussion, it seems arbitrary to call dex the “primary” anesthetic agent in these studies. The study showed that combining sub-anesthetic doses of dex and conventional anesthetics is sufficient to produce surgical anesthesia, so in my view, neither is the “primary” anesthetic.

**********

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Reviewer #1: Yes: Timothy Angelotti MD PhD

Reviewer #2: No

**********

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PLoS One. 2023 Sep 26;18(9):e0291827. doi: 10.1371/journal.pone.0291827.r002

Author response to Decision Letter 0

18 Jul 2023

PONE-D-23-07486

Towards A Potent and Rapidly Reversible Dexmedetomidine-Based General Anesthetic

July 18, 2023

Dear Dr Fiorelli,

We would like to thank the reviewers and the editor for their excellent comments. The changes made in response to the reviews have undoubtedly improved the manuscript.

This letter includes a point-by-point response to each of the comments made by the reviewers and the editor. The reviewer’s abbreviated comments are in bold italics followed immediately by our reply in regular font. The line numbers in the reply to the reviewers correspond to the line numbers in the version with “Tracked Changes – Simple Markup.”

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

We have followed the PLOS One style requirements including file naming conventions.

2. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist.

The Full Arrive 2.0 Guidelines Checklist has been included with the manuscript resubmission.

3. Thank you for stating the following financial disclosure:

"This study is supported by a NIH grant (GM-116119) To ZX and APF."

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This amended role for the funders is accurate and it has been included in the Cover Letter.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

The manuscript has been edited to make it technically sound and to have conclusions supported by the data.

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

The statistics have been redone in a manner that will make it easier to follow.

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

No changes were made to the data underlying the findings with the exception that the data from new figures is now included.

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

The revised manuscript has been comprehensively edited as can be seen by the “Tracked Changes” version. Due to the significant revision, it may be easier to read the untracked version. We hope that the current version is intelligible.

________________________________________

The manuscript was revised to make it more transparent and easier to read. In the earlier version of the paper, we showed that supplementing Dex with propofol, isoflurane, sevoflurane or N2O all produced a potent anesthetic. In the revised study, we have taken isoflurane and N2O out of the study and they will be used in a future publication, as suggested by the reviewer. Propofol and sevoflurane, the two most popular anesthetics in the clinics, now represent the exclusive focus of the revised manuscript. We believe it important to show that both intravenous and inhalational anesthetics can be used to supplement Dex.

The reviewer wrote that “It appears that the authors wanted to test whether the addition of DEX to a standard general anesthetic can reduce the dose of general anesthetic required.”. This comment is the key to our study since other studies have already shown that Dex could reduce the doses needed to produce anesthesia for standard general anesthetics, like sevoflurane in animals and humans. Our approach is different. Our main aim in this study was to use Dex as the primary agent to produce a target specific anesthesia. We tested high dose Dex by itself. Unfortunately, Dex alone did not completely suppress responses to noxious stimuli, nor was it completely reversible. We then assessed whether low dose propofol or sevoflurane converted a more modest dose of Dex into an anesthetic. In our study the primary agent was Dex, since the goal all along was to minimize propofol or sevoflurane. Propofol was used at doses which did not produce a loss of the righting reflex and sevoflurane at a dose that were less than ½ a MAC. At this dose of sevoflurane, all rats reacted strongly to the tail clamp stimuli (Stable 1) and some rats recovered their righting reflex. We designed the experiments to test this Dex based anesthetic strategy and found that it produced a state of general anesthesia appropriate for surgery. Finally, we tested whether the Dex based anesthetic combinations could be reversed in rats using low dose atipamezole and caffeine. It was very effective. This may potentially allow us, or others, to translate such an anesthetic strategy in the future to the human population since none of the drugs were used at doses that should elicit significant unwanted effects.

Tables 1A/1B should be moved to Supplemental Data. It is background information that could be moved to decrease the size of the paper.

Table 1B (isoflurane) no longer pertains to the revised manuscript and has been removed from the manuscript. Table 1A has been moved to supplemental data as requested (it has become STable 1).

The revised manuscript revisits this issue. We took the differences of MACs between our study and others in the literature seriously. We applied the tail clamp at the position of the tails and the duration consistently. We were confident of the gas concentrations since we employed a gas analyzer for every experiment. Even so, the setup was calibrated on two separate occasions. It was properly calibrated. Some of the difference may be due to the strength of noxious stimulus used between our study and the studies by others. More likely the main difference between the MAC values in the literature and those we report in the manuscript is due to the difference in the sampling sites. In our study, a nose cone was connected to the outlet port of the gas chamber by a short length of corrugated tubing. The gas concentration was sampled at the inlet to the corrugated tubing by a gas analyzer and are equivalent to measurements in a sealed gas chamber and alveolar gas concentrations, as were done by others. See the manuscript by White et al showing the difference between different kinds of measurements, in that case between an anesthesia box and alveolar gas at the trach site [1]. In those studies, the concentrations for MAC were measured in the sealed gas chamber or the end-tidal concentrations sampled at the connector to the trachea. Our measurement was only meant to confirm the EC50 or MAC equivalence of our experimental system, not to redefine the MAC for Sevoflurane. This discrepancy is now covered on lines 370-380 (untracked version).

In the revised manuscript we explain that when Dex was supplemented with propofol the rats lost all response to the noxious tail clamp or the surgical incision at propofol doses too low to elicit unconsciousness when used by itself. If we defined these doses as “high dose propofol,” it would be confusing This issue is discussed on lines 288 and 298 (untracked version).

was considered to be low dose. In essence, two “low doses” of propofol were studied.

The information is now presented in a truncated and clearer manner. The revised manuscript explains that we were trying to identify the minimum dose of propofol that when combined with Dex, suppressed all response to a noxious stimulus. For this study, we started with 200 µg/kg/min of propofol. When that proved to be an insufficient dose, the dose was increased to 300 µg/kg/min. The latter dose was enough propofol to suppress all responses to tail clamp in both female and male rats. This is explained on lines 312 to 332 of the revised manuscript (unmarked version). That is why there are two doses of propofol. The statistics have been edited to make them clearer. Instead of describing each time point in detail, the revised manuscript only describes the aggregated data from all the time points (see the paragraph starting at line 312). Nonetheless, Tables 2 and 3 still contain all the statistics for each time point for any interested reader.

If the authors wish to study reversal of DEX effects, it should not have been included in the same study as examining “low dose” propofol. This idea should have been tested separately.

We recently published a study showing that atipamezole and caffeine when used together, rapidly and completely reversed Dex sedation [2]. In that study, we reported lower doses of Atipamezole (1/10 or 1/20 of the manufacturer’s recommended dose) could reverse Dex’s sedative effect rapidly when it was combined with caffeine in rats. If that strategy can be translated to humans, then low dose Atipamezole supplemented with caffeine has the potential to overcome the high dose requirement of Atipamezole thereby minimizing, or even eliminating, the unwanted effects of high dose Atipamezole. We do not have any human data for low dose Atipamezole with caffeine. Even so, we believe that the concept of using low dose Atipamezole with caffeine to be promising. In the current study we supplemented Dex with a low dose of a second agent to create a potent anesthetic, which produced immobility, antinociception, and potentially interfered with memory. Without a reversal agent for these Dex based combinations, the utility in humans would be in doubt. We evaluated atipamezole and caffeine to ensure that it was still capable of reversing Dex with the second agent. It worked perfectly. Dex supplemented with a low dose of a second agent represents a rapidly reversible potent anesthetic. To us that is part of the same story; a drug combination that may be safer than currently employed anesthetics, and which is reversible within seconds. We believe that this is worth reporting the Dex based anesthesia and its reversal together.

Atipamezole was originally evaluated for use in the human population over 30 years ago. For 30 years it has not been employed in humans due to its unfortunate side effect profile at the high doses required to reverse Dex. By adding caffeine, dosages 20-fold lower of atipamezole effectively reversed Dex compared to the doses needed when it is used by itself.

For 30 years no one used atipamezole in humans; the drug was a failure in human medicine. We do not claim that atipamezole is a novel reversal agent. We show a novel strategy for using it that should work in humans as well as rodents. Without effective reversal, expanding the reach of Dex may be problematic. Emergence times are just too slow. That is why we believe that it is important that the reversal data remain in the manuscript. See lines 471-481 of the discussion.

As noted above in the comments to the editor, we removed two anesthetics (isoflurane and N20) to make the paper more accessible, as requested by the reviewer. While we showed the abdominal surgeries were successfully performed under Dex with low propofol. We do not yet know which combination will prove to be the “safest.” That will require additional testing. See line 587 in the Discussion.

Thank you for the suggestion. It made the flow better. The Results section of the manuscript starts with the comparison between low and high dose Dex, as requested. See the beginning of the Results section starting with line 245.

We had the same discussion amongst ourselves. We thought that working anesthesiologists would not necessarily appreciate the pain involved in a noxious tail clamp. They would only believe that we observed immobility if and only if it took place during a painful surgery, as that type of procedure remains the gold standard for a successful anesthetic. Abdominal surgery represents such a painful surgery. We agree with the reviewer that lack of change in vital signs alone is not the proof of a complete anesthetic. While the animals were unconscious, the lack of vital sign change during tail clamp or surgery may represent the antinociceptive property of anesthetics. We discuss why surgery is part of the revised manuscript on line 411 of the Results section. As the reviewer suggested, we only show one anesthetic combination for the surgery in the figure to reduce the size of the MS.

The isoflurane and N2O data were removed, which shortened the manuscript and reduced the number of figures. In the past, our studies were criticized because they did not contain EEG data which precluded exploration of certain kinds of mechanism. We are sure that if we remove the EEG data, that EEG researchers will critique the study. The predominant presence of slow wave (delta, 0.5-4 Hz) is a consistent feature in the surgical state of general anesthesia [3] . Without the ability to directly measure amnesia and awareness in rats, we used the EEG recorded at 1 MAC of sevoflurane or isoflurane as a reference, since these volatile agents ~1 MAC are thought to reach EC100 for amnesia and awareness. The EEG data allowed us to confirm the power of the slow wave in Dex based anesthesia and allowed a comparison of the EEGs between the volatile agents at 1 MAC and the Dex based anesthetic. The persistent presence of the slow wave in EEG further confirmed these animals were in the stage of general anesthesia when they were exposed to the Dex based anesthetic. More importantly, our EEG data suggests that memory may be impaired when Dex is supplemented with either propofol or sevoflurane, a requirement for a general anesthetic. However, the final confirmation of memory under these Dex combinations can only be determined in humans. Because it is in a separate section of the Results section, we feel we should provide the basic pattern of EEG for readers who are interested in the EEG information. Anyone else can simply skip over this section.

In the revised Methods, the rationale for the protocol employed is presented in more detail (see line 167). For example, we used isoflurane simply to render rats’ unconscious such that they could be weighed and an I-V line inserted. In some experiments the EEG electrodes were inserted, and the EEG was recorded at ~ 1 MAC for the comparison under isoflurane. We waited for the isoflurane to wash out before starting our studies of tail clamp and vital signs. In the sevoflurane experiments we used sevoflurane throughout, but it is 10X more expensive than isoflurane, so it was not used in all experiments.

The revised manuscript has been normalized in terms of drug dosages. In the revised manuscript there are no drugs given at 30 minutes. Only at 60 minutes.

In our response to comment #3, above, we explained that we tried to optimize Dex and propofol to achieve the best possible effect. We had two different goals. One was to completely suppress any response to a noxious stimulus, while the second was to minimize the Atipamezole levels needed for complete and rapid reversal of anesthesia. We could achieve EC100 in preventing response to tail clamp stimuli by using Dex at 12 mcg/kg/hr with propofol at 300 mcg/kg/min. We used these dosages for the rest of the study, which also allowed us to lower Atipamezole levels.

In our previous study, we focused mainly on the effect of low Atipamezole with caffeine to reverse the sedative effect of a single dose of Dex and a protocol mimicking Dex sedation for MRI. In this study, we aimed to create an anesthetic strategy which is more target specific and reversible. Emergence from Dex sedation can be slow. Will the recovery of Dex based anesthesia be even slower but still reversible? For example, the rats took ~1 hour to “wake” from Dex supplemented with propofol. And then they were sluggish for an extended duration. Without active reversal it is possible that no one will want to use a Dex based anesthetic even if it is safer. The fact that supplemented Dex can be reversed is an important part of the anesthetic story. We have tried to make this point clearer in the revised Discussion (see line 574). The previous study showed that atipamezole and caffeine reversed Dex by itself. Without the new data contained in this manuscript, everyone would be left wondering whether atipamezole and caffeine also reversed the general anesthetic effects of Dex with propofol or Dex with sevoflurane.

Thank you for this comment. We did a poor job explaining the novelty of the study. It is important to us that our manuscript stand on its own. We need to get this explanation right.

Volatile anesthetics and propofol have been linked to developmental abnormalities in neonatal animals, and with cognitive issues in elderly patients. It is not yet known if the neonatal studies in animals are relevant to the human population. Even with that caveat, it is likely that if safe and effective general anesthetics were available that did not use substantial amounts of sevoflurane, isoflurane, propofol or ketamine, they would supplant these popular general anesthetics. Dex is a safer agent which is why it is common in pediatric sedation.

The studies using Dex to lower the MAC concentration for sevoflurane are undoubtedly trying to reduce the sevoflurane concentration used in surgeries thereby creating safer anesthetics. Our goals were different, even though it is also about creating a safer anesthetic. We were hoping to eliminate the need for the current generation of anesthetics by employing a high concentration of Dex by itself. Does high dose Dex represent a suitable general anesthetic if it is reversible? Would the animals exhibit extreme bradycardia or hypotension? Could high dose Dex be successfully reversed with atipamezole & caffeine? Unfortunately, these studies did not work out. The good news is that high dose Dex did not produce any more bradycardia or hypotension than did lower doses. Unfortunately, some rats still responded to the tail clamp. And although atipamezole & caffeine re-established the righting reflex quickly, within a minute, the animals were sluggish and barely moved for a long time. Reversal was therefore incomplete. In clinical practice, a higher total dose of Dex means a higher dose of Atipamezole for reversal. Higher doses of Atipamezole are not our intent.

Our next goal was to determine the absolute minimum levels of a second agent (isoflurane, sevoflurane, propofol or N2O) would convert a more modest dose of Dex to an effective anesthetic. One that could then be effectively reversed. We were successful in that goal. The second agents were employed at sub hypnotic levels. See line 508 of the discussion.

We explain in the Discussion the relevance of our study relative to what was done previously. In those earlier studies, the primary agent was sevoflurane. In our study the primary agent was Dex. Only a small amount of sevoflurane or propofol was employed. The neuroapoptosis triggered by general anesthetics should be dose dependent. Using a sub hypnotic dose should be as safe as possible. Especially since we tried to identify the lowest effective dose possible.

Finally, we observed that supplementing Dex with any anesthetic produced an effective anesthetic. We evaluated propofol and sevoflurane (but also isoflurane, and N2O). We made no assumptions about which of these combinations was the safest. Our goal, in future studies, is to determine which of these combinations is the safest. It may not necessarily be Dex with sevoflurane.

Please see Discussion line 581.

What the authors do not discuss completely in my opinion is why their data demonstrates such a major effect of DEX compared to prior work.

We performed additional experiments in male rats for this revision. Data from male rats is now included in the revised manuscript. In our earlier study we saw no clear differences between male and female rat responses to Dex’s sedative effect, although there may have been a trend in the direction of males being less sensitive and we had simply not tested a large enough cohort of rats [2]. In the current study we saw no difference between male and female rats when testing combinations of drugs (see Table 3 which compares data from Male and Female rats and Supplementary Figs 3 & 4). Data from male rats is now covered throughout the manuscript. Examples are found on lines 392 and 398.

The revised manuscript has a short new title. “A novel dosing regimen creates an effective dexmedetomidine based anesthetic.”

The reviewer is correct. Aged animals would be preferable. Nonetheless, working with aged animals is difficult and expensive. Our goal was to find out whether this strategy would work at all before going to the time and expense of studying older animals. In future studies we hope to reproduce these studies in aged animals of both sexes. This is discussed on line 589.

The revised Introduction focuses on the background and rationale for the studies. Some results are included as well.

• Why were only female rats used for the study? The NIH and most journals now require the use of both sexes to account for sex as a biological variable.

The revised manuscript includes data from male rats. As you can see from Table 3 and SFigs 3 & 4, there was no clear difference between the sexes.

• Were the anesthetic exposures conducted in random order?

Drugs were applied in a randomized manner. This is found in the methods on line 245.

• It should be clearly stated in the manuscript that atipamezole is not approved for human use. This greatly limits the translational potential of these results to the clinical setting.

The original manuscript contained that information. So does the revised manuscript, paragraph starting on line 471. Nevertheless, this does not limit the translational potential. Quite the contrary. In previous human trials atipamezole was evaluated at a variety of dosages. At the high dosages required to reverse Dex, it produced significant side effects. Too many for regulatory approval. It was also evaluated at lower dosages. At these dosages (<30 mg) it produced no unwanted effects, but it was not able to reverse Dex. In our studies we are using even lower dosages (0.5-1: 1 ratio) than those that produced no unwanted effects. Nonetheless, the drug effectively reverses Dex. Why? It is the caffeine which increases [cAMP]i countering the Dex’s effect to lower [cAMP]i (Ref 1 in this letter). When used together extremely low doses of atipamezole become effective. We believe that this combination has strong translational potential.

• There are far too many figures. Many of them should be combined.

Some data from the original paper has been removed. Some data has moved to Supplementary material. Several Tables have been combined, as requested. For example, see Table 3 which combines most of the tail clamp data.

The revised manuscript revisits this issue (please also see the response to the first reviewer). We too worried about the differences between our study and values in the literature. Although we were confident of the gas concentrations, since we employ a gas analyzer for every experiment, we still had the setup calibrated on two separate occasions. It was properly calibrated. Some of the difference may be due to the powerful noxious stimulus used. More likely most of the difference between the MAC values in the literature and those we report in the manuscript are due to measurement differences. In our study, a nose cone was connected to the outlet port of the gas chamber by a short length of corrugated tubing. The gas concentration was sampled at the inlet to the corrugated tubing by a gas analyzer and are alveolar gas concentrations, as was done by others. See the manuscript by White et al showing the difference between different kinds of measurements, in that case between an anesthesia box and alveolar gas sampled at the tracheostomy [1]. Our measurement was only meant to confirm the EC50 or MAC equivalence of our experimental system, not to redefine the MAC for Sevoflurane. This discrepancy is now covered on lines 370-380 (untracked version).

This is more than a semantic argument on our part. When used by itself, Dex at high dosages is not enough to produce anesthesia in every animal tested but is enough to produce a deep level of unconsciousness. The only thing that will rouse the rats is a powerful noxious stimulus. Otherwise, the rats are “out.” They do not respond to sound or touch. The levels of sevoflurane or propofol used to supplement the Dex, were not, by themselves, sufficient to elicit unconsciousness or if it did so, any stimulus, such as IV insertion, would rouse the animals.

Our goal was to use a large dose of Dex so that we would not have to use much of the second agent. If the second agents are toxic, then minimizing their dosage represents an important objective. This point is covered in the discussion starting on lines 503-510.

References:

1. White PF, Johnston RR, Eger EI. Determination of Anesthetic Requirement in Rats. Anesthesiology. 1974;40: 52–57. doi:10.1097/00000542-197401000-00012

2. Xie Z, Fox AP. Rapid emergence from dexmedetomidine sedation in Sprague Dawley rats by repurposing an α2-adrenergic receptor competitive antagonist in combination with caffeine. BMC Anesthesiol. 2023;23: 39. doi:10.1186/s12871-023-01986-5

3. Akeju O, Brown EN. Neural oscillations demonstrate that general anesthesia and sedative states are neurophysiologically distinct from sleep. Curr Opin Neurobiol. 2017;44: 178–185. doi:10.1016/j.conb.2017.04.011

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PLoS One. doi: 10.1371/journal.pone.0291827.r003

Decision Letter 1

Silvia Fiorelli

13 Aug 2023

PONE-D-23-07486R1Towards A Potent and Rapidly Reversible Dexmedetomidine-Based General AnestheticPLOS ONE

Dear Dr. Xie,

==============================

ACADEMIC EDITOR:please carefully assess all the reviewers comments

==============================

Please submit your revised manuscript by 13 September 2023. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Silvia Fiorelli

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: In Manuscript PONE-D-23-07486_R1, Xie and colleagues have revised their prior manuscript as per the reviewers’ comments. Overall, I appreciate the amount of work and new experiments performed for this revision. By concentrating only one two general anesthetic agents, sevoflurane (sevo) and propofol (prop), the authors have made the paper more focused and thus their conclusions are more easily appreciated. However, there are still some issues that need to be addressed (without more experiments) as discussed below.

1. The introduction is much improved in flow and focus. It is now clear that the authors are attempting to extend prior research on this topic. Previously, others have shown that co-administration of dexmedetomidine (dex) can reduce the MAC (or concentration) of sevo or propofol necessary to achieve an anesthetic state. Namely, the authors are attempting to discern if higher doses of dex co-administered with sevo, or prop can achieve an anesthetic state while using a sub-anesthetic dose of the same.

By describing why high dose dex alone as an anesthetic agent has failed in the past (e.g., slow emergence, lack of good reversal agents, vital sign effects), it becomes more apparent why further work is needed. However, I still struggle with the idea that the combination of dex with other agents can lead to dex being the “primary” agent. For example, prior work has shown that dex can reduce the MAC of another agent. In this work, the authors have also shown that dex can reduce the MAC of another agent, possibly reducing the second agent (sevo or prop) to a concentration/dose that is now considered sub-anesthetic. In other words, the authors appear to not be doing something unique but rather extend prior work to see if the MAC lowering effect of dex can be extended to reduce MAC further by using a higher dose of dex than that studied previously.

2. The data reported in this study strongly suggests that the effect of dex to reduce MAC can be seen at higher doses of dex. The study then is extended to demonstrate that the effects of dex/sevo or dex/prop in a rat model of tail clamping (used to simulate surgical stimulation) can be extended to a sham surgery protocol. This protocol was included to assess if the behaviors seen in the rat model are similar to a surgical protocol. As noted by the authors, they could only measure changes in vital signs as a surrogate for anesthetic effects, but the data appears to support their hypothesis

3. One drawback to the use of dex at higher doses is the need for a reversal agent. As there is no reversal agent approved for humans use, the authors attempted to reverse dex with atipamezole and caffeine (as done in their prior published work). It is clear that this combination of reversal agents works.

4. Lastly, in order to determine if the newer combinations of dex with either sevo or propofol produce amnesia, the authors opted to study these anesthetic combinations with EEG analysis. There is a lot of data and I appreciate that the authors feel it is important to include this data due to concerns that potential reviewers may not agree with their findings without evidence of amnesia.

5. Therefore, the current revision is much improved and more focused in design, but not more focused in the writing. The paper contains large sections of discussions of early data. For example, Page 90 Paragraph 2 is very long to simply make the point that dex suppressed responses to noxious stimuli. These discussions and tables (Table 1-3) are not necessary or should be moved to the supplemental data area. The paper is still too long for the conclusions reached and such detail makes reading the paper difficult.

6. If the authors feel the need to retain the section of EEG analysis, it needs to be shortened. Does all of the detail need to be included?

7. On Page 111 Lines 706-717, the authors concisely summarize their work and results. However, this is buried in the discussion. Similarly, the section on EEG effects (Page 112 Lines 721-738 is summarized nicely. This style of writing is appreciated for its ease of readability. The authors should look for other sections that could similarly be reduced.

8. The figure legends often contain extremely long descriptions of the statistics derived from their work. It isn’t necessary and is impossible to read. If the authors feel this data is needed, then it should be in a supplemental figure/table or somehow included in the figure itself. For example, Figure 5 on Page 121 Line 928-961 is impossible to read. Other figure legends are similar.

9. Overall, a great revision but still central message is lost by the length and unnecessary detail included in the manuscript. The story is now clearer but the writing isn’t.

a) Is it possible to use dex as a primary agent (i.e., a higher dose)?

b) Can we overcome limitations of using higher dex doses (no reversal, slow emergence)?

c) Can co-administration of higher doses of dex further lower the amounts of other general anesthetic agents utilized (e.g., sevo or prop) as suggested by prior work?

d) If so, do such combinations of high dex with sevo or prop produce the other aspects of a general anesthetic (i.e., ability to perform surgery, effects on memory/amnesia)?

Reviewer #2: (No Response)

**********

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Reviewer #1: Yes: Timothy Angelotti MD PhD

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PLoS One. 2023 Sep 26;18(9):e0291827. doi: 10.1371/journal.pone.0291827.r004

Author response to Decision Letter 1

18 Aug 2023

PONE-D-23-07486R2

Towards A Potent and Rapidly Reversible Dexmedetomidine-Based General Anesthetic

August 18, 2023

Dear Dr Fiorelli,

We would like to thank the reviewers for their diligence. Multiple changes were made in response to the reviews to address the comments. Their comments will definitely help to improve our MS.

This letter includes a point-by-point response to each of the comments made by the reviewers and the editor. The reviewer’s comments are in bold italics followed immediately by our reply in regular font. The line numbers in the reply to the reviewers correspond to the line numbers in the version with “Tracked Changes – Simple Markup.”

Thank you for your thoughtful comments. The reviewer correctly pointed out that “The authors are attempting to discern if higher doses of Dex co-administered with sevo, or prop can achieve an anesthetic state while using a sub-anesthetic dose of the same.” The reviewer is also correct that “prior work has shown that dex can reduce the MAC of another agent.” Most of the previous studies using Dex to reduce MAC levels were done in humans (Ref 61,63,64). Here is a summary of the previous studies in question: Dex was used an adjunct agent at the doses ranging from 0.3-0.7 mcg/kg/hr which reduced the MAC concentration of sevoflurane. In those studies, the modest dose of Dex that was employed, reduced the MAC of sevoflurane by 20-30%. In one pediatric study, low dose Dex (0.2 mcg/kg/hr), reduced the required sevoflurane dose by 60%. However, these patients also received a caudal block after induction for the procedure, thus obviating the need to provide analgesia (Ref 62). In another study with isoflurane, higher doses of Dex (up to 2.85 mcg/kg/hr) were used in human volunteers. The MAC of isoflurane was reduced by 50%. However, the recovery time was very long (up to 4-6 hours) in those subjects (1, see reference below; We did not cite this article because we focused on sevoflurane and propofol in the MS). In a case report with three patients, higher doses of Dex (5-10 mcg/kg/hr) alone were used in conjunction with local anesthetics (ref 60) for surgery. Dex at these high doses, which were like the Dex dose (12 mcg/kg/hr) used in rats in our study, produced sufficient anesthesia for the procedures without significant hemodynamic compromise in the three patients. Again, their recovery time was about 2 hrs or longer.

Studies of high dose Dex have been hindered in the adult human population for two reasons. First, when Dex is used in the adult population the dosages are typically low. It is employed at dosages in the range 0.2 - 0.7 mcg/kg/hr. These low dosages already produce significant hemodynamic effects. The worry is that going to higher dosages will exacerbate the hemodynamic effects and this concern prevents higher doses being employed in adults. Second, as you can already see from the brief review of the literature that was presented above, higher doses of Dex produce enormously long emergence times in humans. Hours, to many hours is simply not acceptable. No one will use high dose Dex, even if it is demonstrably safer. It’s just not practical.

In rats, our studies show that the hemodynamic effects of Dex saturate at low concentrations. High dose Dex does not produce additional hemodynamic changes compared to low dose Dex. Most importantly, using our reversal agent, we change the emergence times from hours to seconds. Our results suggest that high dose Dex can be used as the primary agent safely and without tying up recovery facilities for hours. We believe that these results may change the way that clinicians look at Dex.

We agree with the reviewer that on one level we could interpret our study as an extension of some of the above previous work by using a higher dose of Dex to permit a further reduction of the required doses of sevoflurane or propofol. However, we would argue instead that our focus driving this work was not so much the reduction of the required quantities of other common anesthetizing agents using Dex as an “adjunct”, but rather the use of minimal amounts of other agents to allow Dex to function as an effective general anesthetic. On the surface, this characterization appears semantically tautological- much like the distinction between characterizing a glass as “half empty” vs “half full”. We would like to appeal to the very different philosophical and perspectival orientations that underlie the use of one description vs the other. Without an effective reversal for Dex, the use of higher doses of Dex will not be feasible clinically. The use of Dex will be limited at 0.2-0.7 mcg/kg/hr which will not produce unconsciousness in humans. It will be only used as the less potent agent of two agents regardless the combinations. In pediatric sedation, Dex is commonly used at 2-3 mcg/kg/hr. At this dose, most patients are deeply sedated or unconscious. At these doses of Dex, high doses of sevo or propofol are not needed. However, this high dose of Dex prolongs recovery if there is no safe and effective reversal. The unique nature of our study is that we can create a reversible anesthetic combination with more selective targets. We believe the higher the dose of sevoflurane or propofol the less selective they are. In this study, we cannot confirm whether higher dose of Dex and lower dose of sevo or propofol are safer in the elderly or less neurotoxic in neonates. We only confirm this approach is feasible and we will assess these combinations to determine their safety, efficiency and neurotoxic profiles in humans and animals in the future.

The reviewer pointed out that any agent could in principle be considered “primary” in our combinations because we require both Dex and the second agent to maintain robust general anesthesia. To justify our characterization, consider the analogy to how one assigns which component in a liquid/liquid solution is the “solvent” (since one could argue that either liquid could be considered a solvent). Conventionally this assignment is made based on the component that is present in greater chemical quantity. Similarly, we call Dex the “primary” agent in our anesthetic combinations because the Dex was present in greater “anesthetizing quantity” than the other agents with which it was paired. By this we mean that while Dex alone at the doses we used was sufficient to keep all rats unconscious for the duration of infusion, the second agents (propofol or sevoflurane) at the doses we used were unable by themselves to maintain unconsciousness and suppress righting in the rats. In addition, Dex was the drug we reversed. The doses of sevo or propofol were so low that no reversal for sevo or propofol was needed in these combinations.

As we extend our findings into human subjects, we will seek to establish an effective and safe dose in humans which is comparable to the doses of Dex in this animal study. We might start, for example, at 0.7-1 mcg/kg/hr. Pediatric sedation has employed doses up to 3 mc/kg/hr safely. Very likely the sweet spot will be somewhere in between. Armed with an effective reversal, if it proves to be effective, we will be empowered to evaluate Dex at doses higher than those we currently use when employing Dex as an adjunctive agent.

Finally, we defined “low dose” dex as 10 mcg/kg bolus and 12 mcg/kg/hr infusion vs “high dose” as 40 mcg/kg and 48 mcg/kg/hr based on the responses to tail clamp stimuli engendered. Dex at 10 mcg/kg and 12 mcg/kg/hr is considered a very high dose in human despite a case report (Ref 62) documenting comparable doses. In rat studies this dose is on the low end. With this low dose, all rats lost their righting reflex and remained unconscious, but not be able to tolerate any noxious stimuli, including IV insertion. In the study of amphetamine and Dex (ref. 77), those authors used a 50 mcg/kg bolus. It is used at even higher doses in veterinary medicine. We attempted to use the lowest dose of Dex which caused a loss of righting reflex to combine with other subanesthetic doses of sevo or propofol to produce reversable general anesthesia.

We have included these points in the discussion section (please see lines 504-531).

Thank you for your understanding.

Thank you very much.

Thank you for your understanding.

We revised the discussions as instructed by the reviewer. The paragraph 2 in page 90 (previous tracked version) was shortened. Tables 1-3 were moved to supplemental data area. We only described the tail clamp information in the result section.

6. If the authors feel the need to retain the section of EEG analysis, it needs to be shortened. Does all of the detail need to be included?

We feel the EEG analysis is critical in establishing some of the conclusions of our manuscript. There is no other way to monitor brain activity and provide a compelling proxy for amnesia/awareness in the context of an ablated righting reflex. With our basic and non-invasive recording, we tried to compare EEGs between the Dex based combinations and the conventional anesthetic agents. We stated our observation without making any conclusive statement on amnesia. We feel some readers may be interested in the EEG information for the Dex based anesthesia. Some evidence suggested EEG monitoring is helpful to prevent overdose of anesthetics in certain vulnerable populations (2, 3, references below). A recent review paper suggested that intraoperative EGG monitoring is helpful to enhance recovery although there is insufficient outcome data (see reference 4 below). In our study, we compare the EEG spectrograms and power spectra with ~ 1 MAC of volatile agent vs Dex combinations and shown that the Burst-Suppression Ratio under ~ 1 MAC of volatile agent vs Dex combinations were very different.

We have shortened the EEG discussion by moving some statistical information to the figure legends. As the reviewer correctly points out, EEG data is complicated. As such we feel compelled to ensure we have provided sufficient information to the readers in the methods and analysis. We ask for the reviewer’s understanding.

Thank you for your suggestion. We revised the discussion section accordingly.

Thank you for the suggestion. We agree the extensive statistical description is not necessary and may cause confusion. We have re-written the figure 3, 5 and 7 legends and made them shorter. We used the number at the point before the application of Dex or Dex based combination as the baseline. We compared the rest of the time points to the baseline. We hope it is easier to read.

9. Overall, a great revision but still central message is lost by the length and unnecessary detail included in the manuscript. The story is now clearer but the writing isn’t.

a) Is it possible to use dex as a primary agent (i.e., a higher dose)?

b) Can we overcome limitations of using higher dex doses (no reversal, slow emergence)?

c) Can co-administration of higher doses of dex further lower the amounts of other general anesthetic agents utilized (e.g., sevo or prop) as suggested by prior work?

d) If so, do such combinations of high dex with sevo or prop produce the other aspects of a general anesthetic (i.e., ability to perform surgery, effects on memory/amnesia)?

We appreciate these suggestions and include all of them in the discussion.

Reviewer #2: (No Response)

Thank you for reviewing our MS.

References:

1. Khan ZP, Munday IT, Jones RM, Thornton C, Mant TG, Amin D. Effects of dexmedetomidine on isoflurane requirements in healthy volunteers.1: Pharmacodynamic and pharmacokinetic interactions Br J Anaesth. 1999; 83:372–80

2. Koch S et al. Perioperative electroencephalogram spectral dynamics related to postoperative delirium in older patients, Anesthesia & Analg. 2021

3. Yuan I et al. Prevalence of isoelectric electroencephalography events in infants and young children undergoing general anesthesia, Anesthesia & Analg. 2021

4. Chan, MTV et al. American Society for Enhanced Recovery and Perioperative Quality Initiative Joint Consensus Statement on the Role of Neuromonitoring in Perioperative Outcomes: Electroencephalography, Anesthesia & Analg. 2020

Respectful

Zheng (Jimmy) Xie, MD, Ph.D, FASA

Professor

Department of Anesthesia and Critical Care

University of Chicago

Attachment

Submitted filename: Point by Point Rebuttal Letter.8.18.23.docx

Click here for additional data file.^{(28KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0291827.r005

Decision Letter 2

Silvia Fiorelli

6 Sep 2023

Towards A Potent and Rapidly Reversible Dexmedetomidine-Based General Anesthetic

PONE-D-23-07486R2

Dear Dr. Xie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Silvia Fiorelli

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Congratulations to the authors and thanks to the reviewers for the suggestions provided which really helped improve the quality of the manuscript

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Timothy Angelotti MD PhD

Reviewer #2: No

**********

PLoS One. doi: 10.1371/journal.pone.0291827.r006

Acceptance letter

Silvia Fiorelli

15 Sep 2023

PONE-D-23-07486R2

Towards a potent and rapidly reversible Dexmedetomidine-based general anesthetic

Dear Dr. Xie:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Silvia Fiorelli

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Checklist. The ARRIVE guidelines 2.0: Author checklist.

(PDF)

Click here for additional data file.^{(109.6KB, pdf)}

S1 Fig. Scalp EEG lead placement.

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S2 Fig. Determining the dose of propofol required to maintain unconsciousness to define a low dose of the drug.

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S1 Table. Comparing responses to a noxious stimulus in female rats exposed to a lower dose of Dex alone with rats administered a higher dose of Dex alone.

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S2 Table. Comparing responses to a noxious stimulus in rats exposed to Dex alone with rats administered Dex and a low dose of propofol.

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S3 Table. Comparing responses to a noxious stimulus in rats exposed to Dex alone with rats administered Dex and a low dose of either sevoflurane or propofol.

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S4 Table. A—determining the concentration of sevoflurane required to prevent half of the responses to a noxious mechanical stimulus.

Details about the stimulus are in the Methods. The green column represents ~1 MAC equivalence concentration or EC₅₀.

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Attachment

Submitted filename: Point by point rebuttal letter - final.7.18.23.docx

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Attachment

Submitted filename: Point by Point Rebuttal Letter.8.18.23.docx

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Towards a potent and rapidly reversible Dexmedetomidine-based general anesthetic

Zheng Xie

Robert Fong

Aaron P Fox

Roles

Abstract

In conclusion

Introduction

Materials and methods

Ethics and animals

Calibrated noxious stimulus

Determination of minimum alveolar concentration (MAC) equivalence of sevoflurane in suppressing tail clamp stimulus

Drugs

Sedation/ Anesthesia

Dex infusion with and without a second agent

Fig 1. Experimental protocol.

Electroencephalogram (EEG) recording

Power spectra

Statistical analysis

Results

Is high dose of Dex an effective anesthetic?

Fig 2. The combination of atipamezole and caffeine dramatically accelerated emergence from the anesthesia produced by a high dose of Dex used by itself.

Fig 3. Rats exposed to high-dose Dex by itself exhibited slowed heart and respiration rates while leaving blood oxygen saturation unaffected.

Dex and low dose propofol

Fig 4. The combination of atipamezole and caffeine dramatically accelerated emergence from the anesthesia produced by Dex supplemented with a low dose of propofol.

Fig 5. Female rats exposed to Dex supplemented with a low dose of propofol exhibited slowed heart and respiration rates while blood oxygen saturation levels were unaffected.

Determination of EC50 or minimum alveolar concentration (MAC) equivalence of sevoflurane

Dex and low dose sevoflurane

Fig 6. The combination of atipamezole and caffeine dramatically accelerated emergence from the anesthesia produced by Dex supplemented with a low dose of sevoflurane.

Fig 7. Rats exposed to Dex supplemented with a low dose of sevoflurane exhibited slowed heart and respiration rates while leaving blood oxygen saturation unaffected.

Abdominal surgery

Fig 8. No change in vital signs observed during surgery for the combination of Dex and low-dose propofol.

EEG analysis: Sevoflurane vs Dex with low sevoflurane, isoflurane vs Dex with low propofol

Fig 9. Comparing EEGs obtained under 3% sevoflurane and under Dex with low dose sevoflurane.

Fig 10. Comparisons EEGs obtained under isoflurane 1.7% and Dex with low dose propofol (300 μg/kg/min).

Discussion

Limitations

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Silvia Fiorelli

Roles

Author response to Decision Letter 0

Decision Letter 1

Silvia Fiorelli

Roles

Author response to Decision Letter 1

Decision Letter 2

Silvia Fiorelli

Roles

Acceptance letter

Silvia Fiorelli

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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