KEY POINTS
Question : What are the interventions of anesthesia-induced cognitive impairment in young mice?Findings : Alpha-2 adrenergic receptor agonist dexmedetomidine and clonidine mitigated the sevoflurane-induced tau phosphorylation, synaptic loss, and cognitive impairment in young mice.Meaning : These findings suggest that α-2 adrenergic receptor may contribute, at least partially, to the sevoflurane-induced tau phosphorylation and cognitive impairment in young mice, and α-2 adrenergic receptor agonist dexmedetomidine and clonidine may mitigate the effects of sevoflurane in young mice. In preclinical studies, anesthetics cause cell death, impair neurogenesis, and inhibit synapse formation in the brain tissues of young mice via impaired neurotrophin signaling, mitochondrial dysfunction, neuroinflammation, and interneuron phenotype changes. (reviewed in reference 1 2 , 3 4–7 8 9 10 11–13
However, the underlying mechanisms through which sevoflurane induces tau phosphorylation and cognitive impairment in young mice remain to be determined. Specifically, the involvement of α-2 adrenergic receptor in the sevoflurane-induced effects in young mice remains unknown. Moreover, whether anesthetic dexmedetomidine can mitigate the sevoflurane-induced tau phosphorylation and cognitive impairment in young mice has not been determined.
Dexmedetomidine, a highly selective α-2 adrenergic receptor agonist, causes sedative effects by binding to α-2 adrenergic receptors in the locus coeruleus14 15 16–19 20
Dexmedetomidine has been reported to induce tau phosphorylation10 , 21 22 23 24 25
Therefore, the primary objective of the current study was to determine whether anesthetic dexmedetomidine can attenuate the sevoflurane-induced tau phosphorylation and cognitive impairment in young mice. The another objective was to assess the role of α-2 adrenergic receptor in the sevoflurane-induced effects in young mice by using the α-2 adrenergic receptor agonists dexmedetomidine14 26 27
We hypothesized that activation of the α-2 adrenergic receptor by dexmedetomidine and clonidine would attenuate sevoflurane-induced tau phosphorylation and cognitive impairment while yohimbine would reverse the attenuation effects of dexmedetomidine and clonidine. These data would implicate that α-2 adrenergic receptors are one of the underlying mechanisms of anesthesia-induced tau phosphorylation and demonstrate potential for targeted interventions for treating and preventing perioperative neurocognitive disorder in young patients.
METHODS
Mice
All experimental procedures involving mice were approved by the Standing Committee on Animals at Massachusetts General Hospital, Boston, MA (protocol number: 2006N000219) and conformed to National Institutes of Health (Bethesda, MD) guidelines. This article was written according to applicable ARRIVE (Animal research: Reporting In Vivo Experiments) guidelines. Efforts were made to minimize the number of mice used in the studies. Adult mice (C57BL/6J) were purchased from Jackson Laboratory (Jackson Laboratory, Bar Harbor, ME). Mice were housed in the Massachusetts General Hospital animal facility. All mice were fed with standard rodent food and water and were housed (5 mice per cage) in a controlled environment at 37 °C with 12-hour light/dark cycles (lights on from 07:00 to 19:00). The day of birth was designated as P0. The young mice were randomly allocated to different groups. Each group included a similar number of female and male mice. We randomly assigned mice to 10 groups: (1) control plus vehicle; (2) control plus dexmedetomidine; (3) control plus clonidine; (4) control plus yohimbine plus dexmedetomidine; (5) control plus yohimbine plus clonidine; (6) sevoflurane plus vehicle; (7) sevoflurane plus dexmedetomidine; (8) sevoflurane plus clonidine; (9) sevoflurane plus yohimbine plus dexmedetomidine; (10) sevoflurane plus yohimbine plus clonidine. Mice did not experience unexpected lethality and were euthanized according to institutional animal care and committee guidelines.
Anesthesia and Treatment
Given the clinical observation that multiple exposures (eg, 3 times) of anesthesia and surgery to children are associated with the increased risk of development of perioperative neurocognitive disorder, we treated the young mice with 3 times of sevoflurane to conceptually mimic the multiple exposures of anesthesia and surgery in children but also determine the role of anesthesia without surgery in the observed neurocognitive disorder in young mice as performed in our previous studies.11–13 , 28 o 2 and Pco 2 in the mice (Supplemental Digital Content, Table 1, https://links.lww.com/AA/D235 11 , 28 29 30 29 11
New Object Recognition Test
The NOR test was performed on P29 and P30. In the first trial, a mouse was placed in a square arena (60 cm × 60 cm × 60 cm, with an even lighting of 20 lux) to habituate for 5 minutes. We used a black container to cover the mouse. Two identical objects (same shape and color) were placed in opposite corners of the upper half of the area and the mouse was then released from the container. We recorded mouse activity and the time spent interacting with the object for 10 minutes. We repeated the first trial 24 hours later, but one of the objects was replaced with a novel object (different shape and color). The interaction time with the novel object (novel time) and familial object (familiar time) was recorded separately. The discrimination index was defined as the ratio of novel time to the novel time plus familial time, which indicated new object recognition ability.
Morris Water Maze
The MWM test was performed as described previously.11 , 28 28
Brain Tissue Harvest, Lysis, and Protein Quantification
Mice were decapitated on P12 or P37, and we harvested the hippocampi for western blot analysis. We homogenized harvested tissues on ice using an immunoprecipitation buffer (M-PER Mammalian Protein Extraction Reagent, Cat# 78501, Thermo Scientific, Waltham, MA) plus protease inhibitor cocktail (Sigma; Cat# 11836170001). The lysates were collected and centrifuged at 4 °C for 20 minutes at 12,000 revolutions per minute (rpm). The total amounts of protein were quantified by bicinchoninic acid protein assay kit (Pierce, Iselin, NJ).
Western Blot Analysis
Total tau was detected by anti-tau 46 antibody (Cat # T9450, 55 kDa, 1:2000, Sigma). AT8 antibody (Tau-PS202/PT205, Cat # MN1020, 55 kDa, 1:500, Thermo Scientific) was used to detect tau phosphorylated at serine 202 and threonine 205 amino acid. PSD-95 antibody (95 kDa, Cat # GB11277, 1:1000; Cell Signaling, Billerica, MA) was used to detect amount of PSD-95. Finally, anti-β-actin antibody was used to detect nontargeted protein β-actin (42 kDa, 1:5000, Sigma, St. Louis, MO) serving as a control for loading differences in total protein. Western blot quantification was performed using the standard methods as described by Xie et al.31
Statistical Analyses
We present data obtained from the western blot, escape latency of MWW and NOR studies as mean ± standard deviation. Data from the platform crossing times of MWM are presented using median and interquartile range. Interaction between time and group factors was determined by a 2-way ANOVA with repeated measurements (Greenhouse-Geisser correction) to analyze the difference in learning curves (based on escape latency) between mice in the control or vehicle group and mice in the sevoflurane anesthesia, dexmedetomidine, or clonidine with and without yohimbine group in the MWM experiments. A 1-way ANOVA with Bonferroni’s multiple comparison test was used to determine the differences among groups in terms of total tau, tau-PS202/PT205, and PSD-95 amounts. Mann-Whitney U test was used to determine the difference between mice in the control or vehicle groups and mice in the sevoflurane anesthesia, dexmedetomidine, or clonidine with and without yohimbine treatment in terms of platform crossing numbers in MWM, and the ratio of novel time to the novel time plus familiar time. We performed these tests based on the distributional assumptions from our previous similar study. There were no missing data for the variables of the MWM (escape latency and platform crossing number) and NOR during the data analysis. P values <.05 were considered statistically significant and the significance testing was 2 tailed. Statistical analysis was conducted using GraphPad Prism software (version 8.0; GraphPad Software, La Jolla, CA). There were 12 mice in each group for the behavioral studies and 6 mice in each group for the western blot study. The sample size was chosen empirically based on the previous studies.28
RESULTS
Dexmedetomidine or Clonidine Mitigates Sevoflurane-Induced Tau Phosphorylation and Sevoflurane-Induced Reduction of PSD-95 in the Hippocampi of Young Mice
Treatment with dexmedetomidine alone did not apparently alter the amount of tau-PS202/tau-PT205 in the hippocampi of young mice; however, sevoflurane increased the amount of tau-PS202/tau-PT205 compared to control. Dexmedetomidine mitigated the sevoflurane-induced increases in tau-PS202/tau-PT205, as evidenced by less visibilities of bands representing the amounts of tau-PS202/tau-PT205 following sevoflurane plus dexmedetomidine treatment. There were no significant differences in β-actin in the hippocampi of young mice among different conditions (Figure 1A ). Quantification of the western blot corroborated these observations (Figure 1B ). One-way ANOVA demonstrated that there was significant difference on the amounts of tau-PS202/tau-PT205 in the hippocampi of young mice (P < .001, Figure 1B ), and dexmedetomidine mitigated the sevoflurane-induced increases in tau-PS202/tau-PT205: 109 ± 25.1% vs 266 ± 53.4%, P < .001 (Bonferroni’s multiple comparison test). Quantitative western blot analysis showed that there were no significant differences in tau among the 4 conditions (P = .383, Figure 1C , D) and that dexmedetomidine mitigated the sevoflurane-induced increases in the ratio of tau-PS202/tau-PT205 to total tau amounts in the hippocampi of young mice: 94 ± 16.3% vs 240 ± 67.8% (P < .001, Figure 1E ).
Figure 1.: Dexmedetomidine attenuates sevoflurane-induced tau phosphorylation and sevoflurane-induced reduction in PSD-95 in the hippocampi of young mice. A, Tau-PS202/tau-PT205 protein in the hippocampi of young mice after sevoflurane and dexmedetomidine treatment. B, Quantification of western blot in (A). C, Total tau protein after various treatments. D, Quantification of western blot in (C). E, Quantification of the western blots in (A) and (C) presented as the ratio of tau-PS202/tau-PT205 to total tau. F, PSD-95 in the hippocampi of young mice at P37 after dexmedetomidine and sevoflurane. G, Quantification of the western blot shown in (F). There were no significant differences in β-actin amounts among these treatments. A 1-way ANOVA with Bonferroni’s multiple comparison test was used to analyze the data. N = 6 in each group. ANOVA indicates analysis of variance; P, postnatal; PSD, postsynaptic density; tau-PS202, tau phosphorylated at serine-202; tau-PT205, tau phosphorylated at threonine 205.
Dexmedetomidine alone did not significantly alter PSD-95 amounts in the hippocampi of young mice. However, sevoflurane decreased PSD-95 amounts compared to control. Dexmedetomidine treatment mitigated the sevoflurane-induced decrease in PSD-95 (Figure 1F ). Quantification of the western blot corroborated these observations that dexmedetomidine mitigated the sevoflurane-induced decrease in PSD-95 amount: 32 ± 12.4% vs 82 ± 6.6% (P = .003, Figure 1F ).
Figure 2.: Clonidine attenuates sevoflurane-induced tau phosphorylation and sevoflurane-induced reduction in PSD-95 in the hippocampi of young mice. A, tau-PS202/tau-PT205 in the hippocampi of the young mice after clonidine and sevoflurane treatments. B, Quantification of the western blot in (A). There were no significant changes of total tau amounts in the different conditions (C and D). E, Quantification of the western blots in (A) and (C) presented as the ratio of tau-PS202/tau-PT205 to total tau. F, PSD-95 in the hippocampi of young mice compared to control at P37 under sevoflurane and clonidine treatment. G, Quantification of the western blot in (F). There were no significant differences in the β-actin amounts among treatments. A 1-way ANOVA with Bonferroni’s multiple comparison test was used to analyze the data. N = 6 in each group. ANOVA indicates analysis of variance; P, postnatal; PSD, postsynaptic density; tau-PS202, tau phosphorylated at serine-202; tau-PT205, tau phosphorylated at threonine 205.
To further determine the involvement of the α-2 adrenergic receptor in sevoflurane-induced tau phosphorylation, we tested whether clonidine, another α-2 adrenergic receptor agonist, could mitigate sevoflurane-induced tau phosphorylation and reduce PSD-95 in the hippocampi of young mice. Quantitative western blot showed that clonidine treatment attenuated the sevoflurane-induced increases in tau-PS202/tau-PT205: 304 ± 43.1% vs 107 ± 7.5% (P < .001, 1-way ANOVA with Bonferroni’s multiple comparison test, Figure 2A , B). There were no significant differences in total tau or β-actin in the hippocampi of young mice among the different conditions (Figure 2C , D). Clonidine mitigated sevoflurane-induced increases in the ratio of tau-PS202/tau-PT205 to total tau: 355 ± 57.6% vs 125 ± 13.5%, P < .001 (Figure 2E ). Quantitative western blot (Figure 2F , G) also showed that sevoflurane anesthesia decreased PSD-95. Clonidine mitigated the sevoflurane-induced decreases in PSD-95: 95 ± 6.4% vs 62 ± 18.4%, P < .001 (Figure 2G ).
Yohimbine Inhibits the Protective Effects of Dexmedetomidine or Clonidine
Sevoflurane plus vehicle or sevoflurane plus yohimbine plus dexmedetomidine induced tau phosphorylation (Figure 3A , B). There were no significant differences in total tau amounts in the hippocampi of the mice following these treatments (Figure 3C , D). Both sevoflurane plus vehicle and sevoflurane plus yohimbine plus dexmedetomidine increased the ratio of tau-PS202/tau-PT205 to total tau in the hippocampi of young mice compared to control plus vehicle or control plus yohimbine plus dexmedetomidine (P < .001, 1-way ANOVA, Figure 3E ). Quantitative western blot showed that both sevoflurane plus vehicle and sevoflurane plus yohimbine plus dexmedetomidine decreased PSD-95 in the hippocampi of mice compared to control plus vehicle or control plus yohimbine plus dexmedetomidine (P < .001, 1-way ANOVA, Figure 3F , G).
Figure 3.: Yohimbine inhibits the protective effects of dexmedetomidine on attenuating sevoflurane-induced tau phosphorylation and reduction in PSD-95 in the hippocampi of young mice. A, Tau-PS202/tau-PT205 in the hippocampi of young mice under various treatment conditions. B, Quantification of the western blot in (A). There were no significant changes in total tau among the different conditions (C and D). E, Quantification of the western blots in (A) and (C) presented as the ratio of tau-PS202/tau-PT205 to total tau in the hippocampi of young mice. F, PSD-95 in the hippocampi of young mice under various treatment conditions. G, Quantification of the western blot in (F). There were no significant differences in the β-actin amounts in the hippocampi of the young mice among the different conditions. A 1-way ANOVA with Bonferroni’s multiple comparison test was used to analyze the data. N = 6 in each group. ANOVA indicates analysis of variance; P, postnatal; PSD, postsynaptic density; tau-PS202, tau phosphorylated at serine-202; tau-PT205, tau phosphorylated at threonine 205.
Figure 4.: Yohimbine inhibits the protective effect of clonidine on attenuating sevoflurane-induced tau phosphorylation and sevoflurane-induced reduction of PSD-95 in the hippocampi of young mice. A, Tau-PS202/tau-PT205 in the hippocampi of young mice after various treatments. B, Quantification of the western blot in (A). There were no significant changes in total tau in the different conditions (C and D). E, Quantification of the western blots in (A) and (C) presented as the ratio of tau-PS202/tau-PT205 to total tau in the hippocampi of young mice. F, PSD-95 in the hippocampi of young mice under various treatment conditions. G, Quantification of the western blot in (F). There were no significant differences in the β-actin amounts in the hippocampi of the young mice among the different conditions. A 1-way ANOVA with Bonferroni’s multiple comparison test was used to analyze the data. N = 6 in each group. ANOVA indicates analysis of variance; P, postnatal; PSD, postsynaptic density; tau-PS202, tau phosphorylated at serine-202; tau-PT205, tau phosphorylated at threonine 205.
Similarly, both sevoflurane plus vehicle and sevoflurane plus yohimbine plus clonidine increased tau-PS202/tau-PT205 amount (Figure 4A , B) but did not change the amount of total tau (Figure 4C , D), increased the ratio of tau-PS202/tau-PT205 to total tau (Figure 4E ), and decreased PSD-95 amount (Figure 4F , G) in the hippocampi of mice compared to control plus vehicle or control plus yohimbine plus clonidine.
Dexmedetomidine and Clonidine Attenuate Sevoflurane-Induced Cognitive Impairment in Young Mice
There was no significant interaction of treatment (vehicle versus dexmedetomidine) and time (day 31–37), and dexmedetomidine did not significantly alter the escape latency of the young mice in the MWM test compared to vehicle (Figure 5A ). Moreover, dexmedetomidine did not significantly change the platform crossing number of the young mice in the MWM test compared to vehicle (Figure 5B ).
Figure 5.: Dexmedetomidine mitigates sevoflurane-induced cognitive impairment in young mice. Escape latency (A) and platform crossing number (B) of the young mice in the MWM test after dexmedetomidine or vehicle. Escape latency (C) and platform crossing number (D) in the MWM test after sevoflurane or vehicle. Escape latency (E) and platform crossing number (F) in the MWM after pretreatment with dexmedetomidine followed by sevoflurane. G, Ratio of the exploring novel object time to the total object time of the NOR test in young mice treated with dexmedetomidine or control. H, Ratio of exploring novel object time to the total object time in NOR test in mice treated with sevoflurane compared to control condition. I, Ratio of exploring novel object time to the total object time in the NOR test compared to control in the young mice pretreated with dexmedetomidine and then sevoflurane. A 2-way ANOVA and Mann-Whitney U test were used to analyze the data. N = 12 in each group. ANOVA indicates analysis of variance; MWM; Morris Water Maze; NOR, New Object Recognition Test; P, postnatal.
Figure 6.: Clonidine mitigates sevoflurane-induced cognitive impairment in young mice. Escape latency (A) and platform crossing number (B) of young mice on the MWM test compared to vehicle after clonidine treatment. Escape latency (C) and platform crossing number (D) on the MWM test after pretreatment with clonidine followed by sevoflurane treatment. E, Ratio of exploring novel object time to the total object time on the NOR test compared to vehicle after clonidine treatment. F, Ratio of exploring novel object time to the total object time on the NOR test compared to control in young mice pretreated with clonidine followed by sevoflurane. A 2-way ANOVA and Mann-Whitney U test were used to analyze the data. N = 12 in each group. ANOVA indicates analysis of variance; MWM; Morris Water Maze; NOR, New Object Recognition Test; P, postnatal.
Sevoflurane did not significantly alter the escape latency of mice in the MWM (Figure 5C ), but sevoflurane decreased the platform crossing number of the young mice in the MWM test compared to control condition (P = .018, Mann-Whitney U test, Figure 5D ). However, sevoflurane anesthesia did not cause cognitive impairment in the mice that received pretreatment with dexmedetomidine (Figure 5E , F). Consistently, sevoflurane anesthesia induced cognitive impairment in the mice in the NOR as evidenced by the decreased ratio of novel time to the novel plus familiar time in young mice following sevoflurane anesthesia compared to control (Figure 5H ). Whereas dexmedetomidine alone did not cause cognitive impairment in the mice (Figure 5G ), dexmedetomidine attenuated the sevoflurane-induced cognitive impairment in the young mice in the NOR (Figure 5I ). Similarly, clonidine attenuated the sevoflurane-induced cognitive impairment indicated by both the MWM test (Figure 6A–D ) and the NOR (Figure 6E , F).
Yohimbine Inhibits the Protective Effects of Dexmedetomidine or Clonidine on Sevoflurane-Induced Cognitive Impairment in Young Mice
There was no interaction of treatment (control plus vehicle versus control plus yohimbine plus dexmedetomidine) and time (P31–P37) on the escape latency of mice in the MWM test (Supplemental Digital Content, Figure 1A, https://links.lww.com/AA/D235 https://links.lww.com/AA/D235 https://links.lww.com/AA/D235 P = .027, Mann-Whitney U test; Supplemental Digital Content, Figure 1D, https://links.lww.com/AA/D235 https://links.lww.com/AA/D235 https://links.lww.com/AA/D235 https://links.lww.com/AA/D235 https://links.lww.com/AA/D235
DISCUSSION
In this proof of concept study, we determined that the α-2 adrenergic receptor contributes to the development of anesthesia-induced tau phosphorylation in young mice and that activation of α-2 adrenergic receptor can attenuate sevoflurane-induced tau phosphorylation and neurocognitive impairment in young mice. These findings have established a system and revealed that α-2 adrenergic receptor could be one of the upstream mechanisms by which sevoflurane induces tau phosphorylation and cognitive impairment in young mice. Moreover, anesthetic dexmedetomidine may mitigate the sevoflurane-induced tau phosphorylation and cognitive impairment in young mice, pending more confirmative studies.
Specifically, the α-2 adrenergic receptor agonists dexmedetomidine (Figure 1 ) and clonidine (Figure 2 ) attenuated the sevoflurane-induced tau phosphorylation and sevoflurane-induced reduction in PSD-95 amount in the hippocampi of young mice at the end of 3 sevoflurane anesthesia exposure periods on P12 or on P37, respectively. Importantly, the α-2 adrenergic receptor antagonist yohimbine mitigated the protective effects of dexmedetomidine (Figure 3 ) and clonidine (Figure 4 ) on sevoflurane-induced tau phosphorylation and reduction in PSD-95. Moreover, dexmedetomidine (Figure 5 ) and clonidine (Figure 6 ) attenuated the sevoflurane-induced cognitive impairment in the young mice tested at P29–P30 (NOR) and P31–P37 (MWM) while yohimbine reversed the protective effects of dexmedetomidine (Supplemental Digital Content, Figure 1, https://links.lww.com/AA/D235 https://links.lww.com/AA/D235
Whittington et al10 10 22
Interestingly, Whittington et al10
Another study by Hu et al23 23
Our study has several limitations. First, we determined the effects of sevoflurane, dexmedetomidine, clonidine, and yohimbine on a group of mixed sex mice, so it is unknown whether these effects are different between females and males. Second, we used tau-PS202/tau-PT205 to determine the role of the α-2 adrenergic receptor on sevoflurane-induced tau phosphorylation based on our previous studies.11
In conclusion, the α-2 adrenergic receptor agonists dexmedetomidine and clonidine attenuated the sevoflurane-induced tau phosphorylation and synaptic loss in the hippocampi of young mice along with sevoflurane-induced cognitive impairment. Moreover, the α-2 adrenergic receptor antagonist yohimbine reversed the protective effects of dexmedetomidine and clonidine. These findings indicate that the α-2 adrenergic receptor may contribute to the development of sevoflurane-induced tau phosphorylation and synaptic loss. Dexmedetomidine could be used to prevent or treat the anesthesia-induced cognitive impairment in young mice. These findings support a need to determine the clinical relevance of these preclinical findings.
DISCLOSURES
Name: Mingyang Sun, MD, PhD.
Contribution: This author helped design and perform the studies.
Conflicts of Interest: None.
Name: Yuanlin Dong, MD, MS.
Contribution: This author helped perform the studies.
Conflicts of Interest: None.
Name: Mengzhu Li, MD, PhD.
Contribution: This author helped perform the studies.
Conflicts of Interest: None.
Name: Yiying Zhang, MD, PhD.
Contribution: This author helped providing critical comments for the studies.
Conflicts of Interest: None.
Name: Feng Liang, MD, PhD.
Contribution: This author helped providing critical comments for the studies.
Conflicts of Interest: None.
Name: Jiaqiang Zhang, MD, PhD.
Contribution: This author helped generate the concept of the studies.
Conflicts of Interest: None.
Name: Sulpicio G. Soriano, MD.
Contribution: This author helped generate the concept of the studies.
Conflicts of Interest: None.
Name: Zhongcong Xie, MD, PhD.
Contribution: This author helped generate the concept of the studies and write the manuscript.
Conflicts of Interest: Z. Xie provides consulting service to Shanghai 9th and 10th hospitals, Baxter (as an invited speaker), and Novartis.
This manuscript was handled by: Gregory J. Crosby, MD.
REFERENCES
1. Vutskits L, Xie Z. Lasting impact of general anaesthesia on the brain: mechanisms and relevance. Nat Rev Neurosci. 2016;17:705–717.
2. Binder LI, Frankfurter A, Rebhun LI. The distribution of tau in the mammalian central nervous system. J Cell Biol. 1985;101:1371–1378.
3. Dujardin S, Colin M, Buée L. Invited review: animal models of tauopathies and their implications for research/translation into the clinic. Neuropathol Appl Neurobiol. 2015;41:59–80.
4. Grundke-Iqbal I, Iqbal K, Quinlan M, Tung YC, Zaidi MS, Wisniewski HM. Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. J Biol Chem. 1986;261:6084–6089.
5. Grundke-Iqbal I, Iqbal K, Tung YC, Quinlan M, Wisniewski HM, Binder LI. Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci U S A. 1986;83:4913–4917.
6. Trojanowski JQ, Lee VM. Paired helical filament tau in Alzheimer’s disease. The kinase connection. Am J Pathol. 1994;144:449–453.
7. Buée L, Bussière T, Buée-Scherrer V, Delacourte A, Hof PR. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res Brain Res Rev. 2000;33:95–130.
8. Le Freche H, Brouillette J, Fernandez-Gomez FJ, et al. Tau phosphorylation and sevoflurane anesthesia: an association to postoperative cognitive impairment. Anesthesiology. 2012;116:779–787.
9. Whittington RA, Virág L, Marcouiller F, et al. Propofol directly increases tau phosphorylation. PLoS One. 2011;6:e16648.
10. Whittington RA, Virág L, Gratuze M, et al. Dexmedetomidine increases tau phosphorylation under normothermic conditions in vivo and in vitro. Neurobiol Aging. 2015;36:2414–2428.
11. Tao G, Zhang J, Zhang L, et al. Sevoflurane induces tau phosphorylation and glycogen synthase kinase 3β activation in young mice. Anesthesiology. 2014;121:510–527.
12. Lu H, Liufu N, Dong Y, et al. Sevoflurane acts on ubiquitination-proteasome pathway to reduce postsynaptic density 95 protein levels in young mice. Anesthesiology. 2017;127:961–975.
13. Xu G, Lu H, Dong Y, et al. Coenzyme Q10 reduces sevoflurane-induced cognitive deficiency in young mice. Br J Anaesth. 2017;119:481–491.
14. Correa-Sales C, Rabin BC, Maze M. A hypnotic response to dexmedetomidine, an alpha 2 agonist, is mediated in the locus coeruleus in rats. Anesthesiology. 1992;76:948–952.
15. Wunsch H, Kahn JM, Kramer AA, et al. Dexmedetomidine in the care of critically ill patients from 2001 to 2007: an observational cohort study. Anesthesiology. 2010;113:386–394.
16. Su X, Meng ZT, Wu XH, et al. Dexmedetomidine for prevention of delirium in elderly patients after non-cardiac surgery: a randomised, double-blind, placebo-controlled trial. Lancet. 2016;388:1893–1902.
17. Riker RR, Shehabi Y, Bokesch PM, et al.; SEDCOM (Safety and Efficacy of Dexmedetomidine Compared With Midazolam) Study Group. Dexmedetomidine vs midazolam for sedation of critically ill patients: a randomized trial. JAMA. 2009;301:489–499.
18. Djaiani G, Silverton N, Fedorko L, et al. Dexmedetomidine versus propofol sedation reduces delirium after cardiac surgery: a randomized controlled trial. Anesthesiology. 2016;124:362–368.
19. Duan X, Coburn M, Rossaint R, Sanders RD, Waesberghe JV, Kowark A. Efficacy of perioperative dexmedetomidine on postoperative delirium: systematic review and meta-analysis with trial sequential analysis of randomised controlled trials. Br J Anaesth. 2018;121:384–397.
20. Deiner S, Luo X, Lin HM, et al.; and the Dexlirium Writing Group. Intraoperative infusion of dexmedetomidine for prevention of postoperative delirium and cognitive dysfunction in elderly patients undergoing major elective noncardiac surgery: a randomized clinical trial. JAMA Surg. 2017;152:e171505.
21. Huang C, Ho YS, Ng OT, Irwin MG, Chang RC, Wong GT. Dexmedetomidine directly increases tau phosphorylation. J Alzheimers Dis. 2015;44:839–850.
22. Liu JR, Yuki K, Baek C, Han XH, Soriano SG. Dexmedetomidine-induced neuroapoptosis is dependent on its cumulative dose. Anesth Analg. 2016;123:1008–1017.
23. Hu J, Vacas S, Feng X, et al. Dexmedetomidine prevents cognitive decline by enhancing resolution of high mobility group box 1 protein-induced inflammation through a vagomimetic action in mice. Anesthesiology. 2018;128:921–931.
24. Shan Y, Sun S, Yang F, Shang N, Liu H. Dexmedetomidine protects the developing rat brain against the neurotoxicity wrought by sevoflurane: role of autophagy and Drp1-Bax signaling. Drug Des Devel Ther. 2018;12:3617–3624.
25. Wang Y, Wu C, Han B, et al. Dexmedetomidine attenuates repeated propofol exposure-induced hippocampal apoptosis, PI3K/Akt/Gsk-3β signaling disruption, and juvenile cognitive deficits in neonatal rats. Mol Med Rep. 2016;14:769–775.
26. Eisenach JC, De Kock M, Klimscha W. alpha(2)-adrenergic agonists for regional anesthesia. A clinical review of clonidine (1984-1995). Anesthesiology. 1996;85:655–674.
27. Farrow S, Mers A, Banta G, Steigerwalt S, Lockette W. Effect of the alpha 2-adrenergic antagonist yohimbine on orthostatic tolerance. Hypertension. 1990;15:877–880.
28. Shen X, Dong Y, Xu Z, et al. Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. Anesthesiology. 2013;118:502–515.
29. Zhang XY, Liu ZM, Wen SH, et al. Dexmedetomidine administration before, but not after, ischemia attenuates intestinal injury induced by intestinal ischemia-reperfusion in rats. Anesthesiology. 2012;116:1035–1046.
30. Guo L, Yu Y, Xin N, Sun J, Chen Y, Yu M. Clonidine protects against neurotoxicity induced by sevoflurane through NF-κB signaling inhibition and proinflammatory cytokine release in rats. J Mol Neurosci. 2018;65:507–513.
31. Xie Z, Culley DJ, Dong Y, et al. The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid beta-protein level in vivo. Ann Neurol. 2008;64:618–627.
